University of Manchester

ORIGAMI: A Software Suite for Activated Ion Mobility Mass Spectrometry (aIM-MS) Applied To Multimeric Protein AssembliesLukasz G. Migas, Aidan P. France, Bruno Bellina and Perdita E. Barran*Michael Barber Centre for Collaborative Mass Spectrometry, School of Chemistry andManchester Institute of BiotechnologyThe University of Manchester, 131 Princess Street, Manchester, M1 7DN, United Kingdom*Correspondence and request for materials should be addressed to Professor Perdita Barran (email: perdita.barran@manchester.ac.uk)AbstractWe present here a software suite (ORIGAMI) that facilitates the rapid acquisition and analysis of ion mobility data following collisional activation. ORIGAMI was developed for use on Waters Synapt instruments where data acquisition is achieved by interfacing WREnS (Waters Research Enabled Software) and MassLynx. Two components are presented, the first is ORIGAMIMS which enables the activation of ions via a sequential ramping of collision voltage prior to ion mobility analysis. We demonstrate the use of ORIGAMI on the tetrameric assemblies formed by the proteins concanavalin A (103 kDa) and alcohol dehydrogenase (143 kDa). Activation is performed in the trap collision cell of the Synapt TriWave assembly where the collision voltage can be ramped from 0-200 V. All of the acquired data is recorded in a single file which simplifies data acquisition while keeping the acquisition times at approx. 25 minutes for a single charge state exposed to a collision voltage range of 0-200V. Following acquisition, the data are analysed in the second component, ORIGAMIANALYSE, which allows the user to visualise the effect of activation on the mobility of the parent ion, as well as on any fragment ion. The user can export the data in the form of heat maps, waterfall or wire plots as well as interactive and shareable webpages.. In addition, tools implemented in ORIGAMI enable easy data extraction from single or multiple MassLynx .raw files, in-depth interrogation of large datasets, statistical analysis and figure creation capabilities. HighlightsNew methodology for controlling acquisition parameters and faster data analysis following activation of ions separated by ion mobility mass spectrometry.Software package capable of simultaneous analysis of multiple MassLynx .raw files.Visualization of the change in mobilities for both parent and product ions following activation.Easy extraction, data processing and extensive plotting tools. Keywords: Collision Induced Dissociation; CID; Collision Induced Unfolding; CIU; activated Ion Mobility; aIM; Ion mobility; IM; Mass Spectrometry; MS; Software; Python.IntroductionMass spectrometry (MS) and ion mobility mass spectrometry (IM-MS) are commonly used to characterize small molecules,ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1039/C5AN00411J", "ISSN" : "0003-2654", "author" : [ { "dropping-particle" : "", "family" : "Lapthorn", "given" : "Cris", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pullen", "given" : "Frank S.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chowdhry", "given" : "Babur Z.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wright", "given" : "Patricia", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Perkins", "given" : "George L.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Heredia", "given" : "Yanira", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "The Analyst", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2015" ] ] }, "page" : "6814-6823", "publisher" : "Royal Society of Chemistry", "title" : "How useful is molecular modelling in combination with ion mobility mass spectrometry for \u2018small molecule\u2019 ion mobility collision cross-sections?", "type" : "article-journal", "volume" : "140" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1021/jacs.5b01338", "ISBN" : "1520-5126 (Electronic)\\r0002-7863 (Linking)", "ISSN" : "15205126", "PMID" : "25760934", "abstract" : "The immediate environment of a molecule can have a profound influence on its properties. Benzocaine, the ethyl ester of para-aminobenzoic acid that finds an application as a local anesthetic, is found to adopt in its protonated form at least two populations of distinct structures in the gas phase, and their relative intensities strongly depend on the properties of the solvent used in the electrospray ionization process. Here, we combine IR-vibrational spectroscopy with ion mobility-mass spectrometry to yield gas-phase IR spectra of simultaneously m/z and drift-time-resolved species of benzocaine. The results allow for an unambiguous identification of two protomeric species: the N- and O-protonated forms. Density functional theory calculations link these structures to the most stable solution and gas-phase structures, respectively, with the electric properties of the surrounding medium being the main determinant for the preferred protonation site. The fact that the N-protonated form of benzocaine can be found in the gas phase is owed to kinetic trapping of the solution-phase structure during transfer into the experimental setup. These observations confirm earlier studies on similar molecules where N- and O-protonation have been suggested.", "author" : [ { "dropping-particle" : "", "family" : "Warnke", "given" : "Stephan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Seo", "given" : "Jongcheol", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Boschmans", "given" : "Jasper", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sobott", "given" : "Frank", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Scrivens", "given" : "James H.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bleiholder", "given" : "Christian", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bowers", "given" : "Michael T.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gewinner", "given" : "Sandy", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sch\u00f6llkopf", "given" : "Wieland", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pagel", "given" : "Kevin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Helden", "given" : "Gert", "non-dropping-particle" : "Von", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of the American Chemical Society", "id" : "ITEM-2", "issue" : "12", "issued" : { "date-parts" : [ [ "2015" ] ] }, "page" : "4236-4242", "title" : "Protomers of benzocaine: Solvent and permittivity dependence", "type" : "article-journal", "volume" : "137" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "1,2", "plainTextFormattedCitation" : "1,2", "previouslyFormattedCitation" : "1,2" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }1,2 peptides,ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/jacs.6b04550", "ISSN" : "0002-7863", "abstract" : "The dynamic nature of intrinsically disordered peptides makes them a challenge to characterize by solution-phase techniques. In order to gain insight into the relation between the disordered state and the environment, we explore the conformational space of the N-terminal 1-5 fragment of bradykinin (BK[1-5]2+) in the gas phase by combining drift tube ion mobility, cold-ion spectroscopy, and first principles simulations. The ion-mobility distribution of BK[1-5]2+ consists of two well-separated peaks. We demonstrate that the conformations within the peak with larger cross-section are kinetically trapped, while the more compact peak contains low-energy structures. This is a result of cis-trans isomerization of the two prolyl-peptide bonds in BK[1-5]2+. Density-functional theory calculations reveal that the compact structures have two very different geometries with cis-trans and trans-cis backbone conformations. Using the experimental CCSs to guide the conformational search, we find that the kinetically trappe...", "author" : [ { "dropping-particle" : "", "family" : "Voronina", "given" : "Liudmila", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Masson", "given" : "Antoine", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kamrath", "given" : "Michael Z.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Schubert", "given" : "Franziska", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Clemmer", "given" : "David E.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Baldauf", "given" : "Carsten", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rizzo", "given" : "Thomas R.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "id" : "ITEM-1", "issue" : "29", "issued" : { "date-parts" : [ [ "2016" ] ] }, "page" : "9224-9233", "title" : "Conformations of prolyl-peptide bonds in the bradykinin 1-5 fragment in solution and in the gas phase", "type" : "article-journal", "volume" : "138" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "3", "plainTextFormattedCitation" : "3", "previouslyFormattedCitation" : "3" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }3 proteinsADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/ncomms12163", "ISBN" : "2041-1723 (Electronic)\\r2041-1723 (Linking)", "ISSN" : "2041-1723", "PMID" : "27418477", "abstract" : "Fdc1 is a decarboxylase enzyme that requires the novel prenylated FMN cofactor for activity. Here, we use it as an exemplar system to show how native top-down and bottom-up mass spectrometry can measure the structural effect of cofactor binding by a protein. For Fdc1Ubix, the cofactor confers structural stability to the enzyme. IM\u2013MS shows the holo protein to exist in four closely related conformational families, the populations of which differ in the apo form; the two smaller families are more populated in the presence of the cofactor and depopulated in its absence. These findings, supported by MD simulations, indicate a more open structure for the apo form. 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IM-MS has been utilised to study the conformational landscapes of mono- and homo-oligomeric proteins,ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "author" : [ { "dropping-particle" : "", "family" : "Clemmer", "given" : "David E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hudgins", "given" : "Robert R", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Jamold", "given" : "Martin F", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "id" : "ITEM-1", "issue" : "18", "issued" : { "date-parts" : [ [ "1995" ] ] }, "page" : "10141-10142", "title" : "Naked Protein Conformations: Cytochrome", "type" : "article-journal" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "6", "plainTextFormattedCitation" : "6", "previouslyFormattedCitation" : "6" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }6 protein-proteinADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/acs.analchem.5b03441", "ISSN" : "0003-2700", "author" : [ { "dropping-particle" : "", "family" : "Quintyn", "given" : "Royston S.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Zhou", "given" : "Mowei", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yan", "given" : "Jing", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wysocki", "given" : "Vicki H.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Analytical Chemistry", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2015" ] ] }, "page" : "acs.analchem.5b03441", "title" : "Surface-Induced Dissociation Mass Spectra as a Tool for Distinguishing Different Structural Forms of Gas-Phase Multimeric Protein Complexes", "type" : "article-journal" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "7", "plainTextFormattedCitation" : "7", "previouslyFormattedCitation" : "7" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }7 and protein-ligand complexes.ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.str.2017.03.005", "ISSN" : "09692126", "author" : [ { "dropping-particle" : "", "family" : "Pacholarz", "given" : "Kamila J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Burnley", "given" : "Rebecca J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Jowitt", "given" : "Thomas A.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ordsmith", "given" : "Victoria", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pisco", "given" : "Jo\u00e3o Pedro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Porrini", "given" : "Massimiliano", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Larrouy-Maumus", "given" : "G\u00e9rald", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Garlish", "given" : "Rachel A.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Taylor", "given" : "Richard J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Carvalho", "given" : "Luiz Pedro S\u00f3rio", "non-dropping-particle" : "de", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Barran", "given" : "Perdita E.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Structure", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2017" ] ] }, "page" : "1-9", "title" : "Hybrid Mass Spectrometry Approaches to Determine How L-Histidine Feedback Regulates the Enzyzme MtATP-Phosphoribosyltransferase", "type" : "article-journal", "volume" : "25" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1038/ncomms12163", "ISBN" : "2041-1723 (Electronic)\\r2041-1723 (Linking)", "ISSN" : "2041-1723", "PMID" : "27418477", "abstract" : "Fdc1 is a decarboxylase enzyme that requires the novel prenylated FMN cofactor for activity. Here, we use it as an exemplar system to show how native top-down and bottom-up mass spectrometry can measure the structural effect of cofactor binding by a protein. For Fdc1Ubix, the cofactor confers structural stability to the enzyme. IM\u2013MS shows the holo protein to exist in four closely related conformational families, the populations of which differ in the apo form; the two smaller families are more populated in the presence of the cofactor and depopulated in its absence. These findings, supported by MD simulations, indicate a more open structure for the apo form. 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Identifying such inhibitors is challenging, however, because they can have Kd values similar to molecules known to inhibit kinase function by interacting with the active form. Further, while inhibitor induced changes within the kinase tertiary structure are significant, few technologies are able to correctly assign inhibitor binding modes in a high-throughput fashion based exclusively on protein-inhibitor complex formation and changes in local protein structure. We have developed a new assay, using ion mobility-mass spectrometry, capable of both rapidly detecting inhibitor binding and classifying the resultant kinase binding modes. Here, we demonstrate the ability of our approach to classify a broad set of kinase inhibitors, using micrograms of protein, without the need for protein modification or tagging.", "author" : [ { "dropping-particle" : "", "family" : "Rabuck", "given" : "Jessica N.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hyung", "given" : "Suk Joon", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ko", "given" : "Kristin S.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fox", "given" : "Christel C.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Soellner", "given" : "Matthew B.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ruotolo", "given" : "Brandon T.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Analytical Chemistry", "id" : "ITEM-3", "issue" : "15", "issued" : { "date-parts" : [ [ "2013" ] ] }, "page" : "6995-7002", "title" : "Activation state-selective kinase inhibitor assay based on ion mobility-mass spectrometry", "type" : "article-journal", "volume" : "85" }, "uris" : [ "" ] }, { "id" : "ITEM-4", "itemData" : { "DOI" : "10.1021/ja306519h", "ISBN" : "1520-5126 (Electronic)\\r0002-7863 (Linking)", "ISSN" : "00027863", "PMID" : "23106332", "abstract" : "The leucine zipper interaction between MAX and c-MYC has been studied using mass spectrometry and drift time ion mobility mass spectrometry (DT IM-MS) in addition to circular dichroism spectroscopy. Peptides comprising the leucine zipper sequence with (c-MYC-Zip residues 402-434) and without a postulated small-molecule binding region (c-MYC-Zip\u0394DT residues 406-434) have been synthesized, along with the corresponding MAX leucine zipper (MAX-Zip residues 74-102). c-MYC-Zip:MAX-Zip complexes are observed both in the absence and in the presence of the reported small-molecule inhibitor 10058-F4 for both forms of c-MYC-Zip. DT IM-MS, in combination with molecular dynamics (MD), shows that the c-MYC-Zip:MAX-Zip complex [M+5H](5+) exists in two conformations, one extended with a collision cross section (CCS) of 1164 \u00b1 9.3 \u00c5(2) and one compact with a CCS of 982 \u00b1 6.6 \u00c5(2); similar values are observed for the two forms of c-MYC-Zip\u0394DT:MAX-Zip. Candidate geometries for the complexes have been evaluated with MD simulations. The helical leucine zipper structure previously determined from NMR measurements (Lavigne, P.; et al. J. Mol. Biol. 1998, 281, 165), altered to include the DT region and subjected to a gas-phase minimization, yields a CCS of 1247 \u00c5(2), which agrees with the extended conformation we observe experimentally. More extensive MD simulations provide compact complexes which are found to be highly disordered, with CCSs that correspond to the compact form from experiment. In the presence of the ligand, the leucine zipper conformation is completely inhibited and only the more disordered species is observed, providing a novel method to study the effect of interactions of disordered systems and subsequent inhibition of the formation of an ordered helical complex.", "author" : [ { "dropping-particle" : "", "family" : "Harvey", "given" : "Sophie R.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Porrini", "given" : "Massimiliano", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Stachl", "given" : "Christiane", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "MacMillan", "given" : "Derek", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Zinzalla", "given" : "Giovanna", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Barran", "given" : "Perdita E.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of the American Chemical Society", "id" : "ITEM-4", "issue" : "47", "issued" : { "date-parts" : [ [ "2012" ] ] }, "page" : "19384-19392", "title" : "Small-molecule inhibition of c-MYC:MAX leucine zipper formation is revealed by ion mobility mass spectrometry", "type" : "article-journal", "volume" : "134" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "4,8\u201310", "plainTextFormattedCitation" : "4,8\u201310", "previouslyFormattedCitation" : "4,8\u201310" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }4,8–10 In addition to gas phase separation, MS and IM-MS has been used to study biophysical phenomena such as gas phase protein rearrangement,ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.str.2017.03.005", "ISSN" : "09692126", "author" : [ { "dropping-particle" : "", "family" : "Pacholarz", "given" : "Kamila J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Burnley", "given" : "Rebecca J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Jowitt", "given" : "Thomas A.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ordsmith", "given" : "Victoria", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pisco", "given" : "Jo\u00e3o Pedro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Porrini", "given" : "Massimiliano", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Larrouy-Maumus", "given" : "G\u00e9rald", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Garlish", "given" : "Rachel A.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Taylor", "given" : "Richard J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Carvalho", "given" : "Luiz Pedro S\u00f3rio", "non-dropping-particle" : "de", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Barran", "given" : "Perdita E.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Structure", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2017" ] ] }, "page" : "1-9", "title" : "Hybrid Mass Spectrometry Approaches to Determine How L-Histidine Feedback Regulates the Enzyzme MtATP-Phosphoribosyltransferase", "type" : "article-journal", "volume" : "25" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "8", "plainTextFormattedCitation" : "8", "previouslyFormattedCitation" : "8" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }8 stabilisation and rearrangement of protein ensembles upon ligand binding,ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "ISSN" : "0306-0012", "abstract" : "The initial stages of drug discovery are increasingly reliant on development and improvement of analytical methods to investigate protein-protein and protein-ligand interactions. For over 20 years, mass spectrometry (MS) has been recognized as providing a fast, sensitive and high-throughput methodology for analysis of weak non-covalent complexes. Careful control of electrospray ionization conditions has enabled investigation of the structure, stability and interactions of proteins and peptides in a solvent free environment. This critical review covers the use of mass spectrometry for kinetic, dynamic and structural studies of proteins and protein complexes. We discuss how conjunction of mass spectrometry with related techniques and methodologies such as ion mobility, hydrogen-deuterium exchange (HDX), protein footprinting or chemical cross-linking can provide us with structural information useful for drug development. Along with other biophysical techniques, such as NMR or X-ray crystallography, mass spectrometry provides a powerful toolbox for investigation of biological problems of medical relevance (204 references).", "author" : [ { "dropping-particle" : "", "family" : "Pacholarz", "given" : "K J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Garlish", "given" : "R A", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Taylor", "given" : "R J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Barran", "given" : "P E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Chemical Society Reviews", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2012" ] ] }, "language" : "English", "note" : "941wv\nTimes Cited:31\nCited References Count:205", "page" : "4335-4355", "publisher-place" : "Barran, PE Univ Edinburgh, Sch Chem, W Mains Rd, Edinburgh EH9 3JJ, Midlothian, Scotland Univ Edinburgh, Sch Chem, Edinburgh EH9 3JJ, Midlothian, Scotland UCB Celltech, Slough SL1 4EN, Berks, England", "title" : "Mass spectrometry based tools to investigate protein-ligand interactions for drug discovery", "type" : "article-journal", "volume" : "41" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1016/j.str.2017.03.005", "ISSN" : "09692126", "author" : [ { "dropping-particle" : "", "family" : "Pacholarz", "given" : "Kamila J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Burnley", "given" : "Rebecca J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Jowitt", "given" : "Thomas A.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ordsmith", "given" : "Victoria", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pisco", "given" : "Jo\u00e3o Pedro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Porrini", "given" : "Massimiliano", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Larrouy-Maumus", "given" : "G\u00e9rald", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Garlish", "given" : "Rachel A.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Taylor", "given" : "Richard J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Carvalho", "given" : "Luiz Pedro S\u00f3rio", "non-dropping-particle" : "de", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Barran", "given" : "Perdita E.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Structure", "id" : "ITEM-2", "issued" : { "date-parts" : [ [ "2017" ] ] }, "page" : "1-9", "title" : "Hybrid Mass Spectrometry Approaches to Determine How L-Histidine Feedback Regulates the Enzyzme MtATP-Phosphoribosyltransferase", "type" : "article-journal", "volume" : "25" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "8,11", "plainTextFormattedCitation" : "8,11", "previouslyFormattedCitation" : "8,11" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }8,11 proton- and electron transfer mechanismsADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.ijms.2016.08.006", "ISSN" : "13873806", "abstract" : "A novel mass spectrometry approach is reported which investigate how ion-molecule charge reduction reactions between radical anions and protein cations modulate protein conformation. An electron transfer reagent (1,3-dicyanobenzene) transfers electrons to positively charged proteins and there are no observable products of dissociation. ETnoD product ions are detected as charged-reduced species with the same molecular weight as the precursor ion, and no significant evidence for proton transfer. We present collision cross section distributions of precursor and product ions before and after exposure to radical ions. Cytochrome c and myoglobin are examined as exemplar systems under both aqueous salt and denaturing conditions before and after exposure to radical anions. We consistently observe depletion of the more compact precursor ion conformers on exposure to the ETD reagent. Remarkably, by examining the collision cross section distributions of the product ions it can be seen that the addition of a single electron can cause a dramatic rearrangement in protein conformation for charge states that are highly populated when sprayed from salty aqueous conditions. Furthermore, a given net charge on an exposed precursor and product ion favours a preferred collision cross section distribution, indicating that the distribution of charge on proteins in the gas phase dictate their conformation. An exception is reported for the low charge state of cytochrome c where compaction was seen in the radical formed post reduction compared to the electrospray generated ion under ETnoD optimised conditions. We propose a model that postulates how electron transfer to conformation stabilising salt bridges may explain our observations.", "author" : [ { "dropping-particle" : "", "family" : "Jhingree", "given" : "Jacquelyn R.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Beveridge", "given" : "Rebecca", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Dickinson", "given" : "Eleanor R.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Williams", "given" : "Jonathan P.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Brown", "given" : "Jeffery M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bellina", "given" : "Bruno", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Barran", "given" : "Perdita E.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "International Journal of Mass Spectrometry", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2016" ] ] }, "publisher" : "Elsevier B.V.", "title" : "Electron transfer with no dissociation ion mobility\u2013mass spectrometry (ETnoD IM\u2013MS). The effect of charge reduction on protein conformation", "type" : "article-journal" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1021/jacs.6b04282", "ISSN" : "15205126", "PMID" : "27399988", "abstract" : "The structure and folding of a protein in solution depends on noncovalent interactions within the protein and those with surrounding ions and molecules. Decoupling these interactions in solution is challenging, which has hindered the development of accurate physics-based models for structure prediction. Investigations of proteins in the gas phase can be used to selectively decouple factors affecting the structures of proteins. Here, we use cation-to-anion proton-transfer reactions (CAPTR) to reduce the charge states of denatured ubiquitin ions in the gas phase, and ion mobility to probe their structures. In CAPTR, a precursor charge state is selected (P) and reacted with monoanions to generate charge-reduced product ions (C). Following each CAPTR event, denatured ubiquitin ions (13+ to 6+) yield products that rapidly isomerize to structures that have smaller collision cross sections (\u03a9). The \u03a9 values of CAPTR product ions depend strongly on C and very weakly on P. Pre- and post-CAPTR activation was then u...", "author" : [ { "dropping-particle" : "", "family" : "Laszlo", "given" : "Kenneth J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Munger", "given" : "Eleanor B.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bush", "given" : "Matthew F.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of the American Chemical Society", "id" : "ITEM-2", "issue" : "30", "issued" : { "date-parts" : [ [ "2016" ] ] }, "page" : "9581-9588", "title" : "Folding of Protein Ions in the Gas Phase after Cation-to-Anion Proton-Transfer Reactions", "type" : "article-journal", "volume" : "138" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "12,13", "plainTextFormattedCitation" : "12,13", "previouslyFormattedCitation" : "12,13" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }12,13 and protein unfoldingADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/ncomms12163", "ISBN" : "2041-1723 (Electronic)\\r2041-1723 (Linking)", "ISSN" : "2041-1723", "PMID" : "27418477", "abstract" : "Fdc1 is a decarboxylase enzyme that requires the novel prenylated FMN cofactor for activity. Here, we use it as an exemplar system to show how native top-down and bottom-up mass spectrometry can measure the structural effect of cofactor binding by a protein. For Fdc1Ubix, the cofactor confers structural stability to the enzyme. IM\u2013MS shows the holo protein to exist in four closely related conformational families, the populations of which differ in the apo form; the two smaller families are more populated in the presence of the cofactor and depopulated in its absence. These findings, supported by MD simulations, indicate a more open structure for the apo form. HDX-MS reveals that while the dominant structural changes occur proximal to the cofactor-binding site, rearrangements on cofactor binding are evident throughout the protein, predominantly attributable to allosteric conformational tightening, consistent with IM\u2013MS data.", "author" : [ { "dropping-particle" : "", "family" : "Beveridge", "given" : "Rebecca", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Migas", "given" : "Lukasz G.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Payne", "given" : "Karl A. P.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Scrutton", "given" : "Nigel S.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Leys", "given" : "David", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Barran", "given" : "Perdita E.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature Communications", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2016" ] ] }, "page" : "12163", "publisher" : "Nature Publishing Group", "title" : "Mass spectrometry locates local and allosteric conformational changes that occur on cofactor binding", "type" : "article-journal", "volume" : "7" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1016/j.jasms.2009.06.010", "ISSN" : "1879-1123", "PMID" : "19643633", "abstract" : "Ion mobility spectrometry, with subsequent mass spectrometric detection, has been employed to study the stability of compact protein conformations of FK-binding protein, hen egg-white lysozyme, and horse heart myoglobin in the presence and absence of bound ligands. Protein ions, generated by electrospray ionization from ammonium acetate buffer, were activated by collision with argon gas to induce unfolding of their compact structures. The collisional cross sections (Omega) of folded and unfolded conformations were measured in the T-Wave mobility cell of a Waters Synapt HDMS (Waters, Altrincham, UK) employing a calibration against literature values for a range of protein standards. In the absence of activation, collisional cross section measurements were found to be consistent with those predicted for folded protein structures. Under conditions of defined collisional activation energies partially unfolded conformations were produced. The degree of unfolding and dissociation induced by these defined collision energies are related to the stability of noncovalent intra- and intermolecular interactions within protein complexes. These findings highlight the additional conformational stability of protein ions in the gas phase resulting from ligand binding.", "author" : [ { "dropping-particle" : "", "family" : "Hopper", "given" : "Jonathan T S", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Oldham", "given" : "Neil J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of the American Society for Mass Spectrometry", "id" : "ITEM-2", "issue" : "10", "issued" : { "date-parts" : [ [ "2009", "10" ] ] }, "page" : "1851-8", "publisher" : "Elsevier Inc.", "title" : "Collision induced unfolding of protein ions in the gas phase studied by ion mobility-mass spectrometry: the effect of ligand binding on conformational stability.", "type" : "article-journal", "volume" : "20" }, "uris" : [ "" ] }, { "id" : "ITEM-3", "itemData" : { "author" : [ { "dropping-particle" : "", "family" : "Tian", "given" : "Yuwei", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ruotolo", "given" : "Brandon T", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "id" : "ITEM-3", "issued" : { "date-parts" : [ [ "2016" ] ] }, "title" : "Ion Mobility-Mass Spectrometry and Collision Induced Unfolding Rapidly Detect Subtle Differences in Antibody Glycoforms Therapeutic Antibodies : Impact and Opportunity", "type" : "article-journal" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "4,14,15", "plainTextFormattedCitation" : "4,14,15", "previouslyFormattedCitation" : "4,14,15" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }4,14,15, with the latter growing in popularity. In IM-MS, conformations are determined by measuring the time it takes ions to cross a mobility device. This data is recorded in the form of an arrival time distribution (ATD) which can then be converted to a rotationally averaged collision cross section (CCS). IM separation is enabled by pulsing packets of ions into a mobility cell filled with an inert buffer gas held under the influence of a weak electric field, the latter serves to propel ions through the cell with a velocity that depends on their charge and on the nature of their collisions with the buffer gas.ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "author" : [ { "dropping-particle" : "", "family" : "Mason", "given" : "E. A.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "McDaniel", "given" : "E. W.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "1988" ] ] }, "publisher" : "Wiley, New York", "title" : "Transport properties of ions in gases", "type" : "book" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1002/rcm.1641", "ISSN" : "0951-4198", "PMID" : "15386629", "abstract" : "The use of radio-frequency (RF)-only ion guides for efficient transport of ions through regions of a mass spectrometer where the background gas pressure is relatively high is widespread in present instrumentation. Whilst multiple collisions between ions and the background gas can be beneficial, for example in inducing fragmentation and/or decreasing the spread in ion energies, the resultant reduction of ion axial velocity can be detrimental in modes of operation where a rapidly changing influx of ions to the gas-filled ion guide needs to be reproduced at the exit. In general, the RF-only ion guides presently in use are based on multipole rod sets. Here we report investigations into a new mode of ion propulsion within an RF ion guide based on a stack of ring electrodes. Ion propulsion is produced by superimposing a voltage pulse on the confining RF of an electrode and then moving the pulse to an adjacent electrode and so on along the guide to provide a travelling voltage wave on which the ions can surf. Through appropriate choice of the travelling wave pulse height, velocity and gas pressure it will be shown that the stacked ring ion guide with the travelling wave is effective as a collision cell in a tandem mass spectrometer where fast mass scanning or switching is required, as an ion mobility separator at pressures around 0.2 mbar, as an ion delivery device for enhancement of duty cycle on an orthogonal acceleration time-of-flight (oa-TOF) mass analyser, and as an ion fragmentation device at higher wave velocities.", "author" : [ { "dropping-particle" : "", "family" : "Giles", "given" : "Kevin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pringle", "given" : "Steven D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Worthington", "given" : "Kenneth R", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Little", "given" : "David", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wildgoose", "given" : "Jason L", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bateman", "given" : "Robert H", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Rapid communications in mass spectrometry : RCM", "id" : "ITEM-2", "issue" : "20", "issued" : { "date-parts" : [ [ "2004", "1" ] ] }, "page" : "2401-2414", "title" : "Applications of a travelling wave-based radio-frequency-only stacked ring ion guide.", "type" : "article-journal", "volume" : "18" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "16,17", "plainTextFormattedCitation" : "16,17", "previouslyFormattedCitation" : "16,17" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }16,17 Compact ions undergo fewer collisions with the gas and will exit the cell earlier than more extended forms. Presently, the majority of IM-MS research on biomolecular complexes is carried out on Travelling-Wave IM-MS (TWIM-MS) Waters SynaptADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1002/rcm.1641", "ISSN" : "0951-4198", "PMID" : "15386629", "abstract" : "The use of radio-frequency (RF)-only ion guides for efficient transport of ions through regions of a mass spectrometer where the background gas pressure is relatively high is widespread in present instrumentation. Whilst multiple collisions between ions and the background gas can be beneficial, for example in inducing fragmentation and/or decreasing the spread in ion energies, the resultant reduction of ion axial velocity can be detrimental in modes of operation where a rapidly changing influx of ions to the gas-filled ion guide needs to be reproduced at the exit. In general, the RF-only ion guides presently in use are based on multipole rod sets. Here we report investigations into a new mode of ion propulsion within an RF ion guide based on a stack of ring electrodes. Ion propulsion is produced by superimposing a voltage pulse on the confining RF of an electrode and then moving the pulse to an adjacent electrode and so on along the guide to provide a travelling voltage wave on which the ions can surf. Through appropriate choice of the travelling wave pulse height, velocity and gas pressure it will be shown that the stacked ring ion guide with the travelling wave is effective as a collision cell in a tandem mass spectrometer where fast mass scanning or switching is required, as an ion mobility separator at pressures around 0.2 mbar, as an ion delivery device for enhancement of duty cycle on an orthogonal acceleration time-of-flight (oa-TOF) mass analyser, and as an ion fragmentation device at higher wave velocities.", "author" : [ { "dropping-particle" : "", "family" : "Giles", "given" : "Kevin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pringle", "given" : "Steven D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Worthington", "given" : "Kenneth R", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Little", "given" : "David", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wildgoose", "given" : "Jason L", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bateman", "given" : "Robert H", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Rapid communications in mass spectrometry : RCM", "id" : "ITEM-1", "issue" : "20", "issued" : { "date-parts" : [ [ "2004", "1" ] ] }, "page" : "2401-2414", "title" : "Applications of a travelling wave-based radio-frequency-only stacked ring ion guide.", "type" : "article-journal", "volume" : "18" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1002/rcm.5013", "ISSN" : "1097-0231", "PMID" : "21594930", "abstract" : "The use of ion mobility separation to determine the collision cross-section of a gas-phase ion can provide valuable structural information. The introduction of travelling-wave ion mobility within a quadrupole/time-of-flight mass spectrometer has afforded routine collision cross-section measurements to be performed on a range of ionic species differing in gas-phase size/structure and molecular weight at physiologically relevant concentrations. Herein we discuss the technical advances in the second-generation travelling-wave ion mobility separator, which result in up to a four-fold increase in mobility resolution. This improvement is demonstrated using two reverse peptides (mw 490 Da), small ruthenium-containing anticancer drugs (mw 427 Da), a cisplatin-modified protein (mw 8776 Da) and the noncovalent tetradecameric chaperone complex GroEL (mw 802 kDa). What is also shown are that the collision cross-sections determined using the second-generation mobility separator correlate well with the previous generation and theoretically derived values.", "author" : [ { "dropping-particle" : "", "family" : "Giles", "given" : "Kevin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Williams", "given" : "Jonathan P", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Campuzano", "given" : "Iain", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Rapid communications in mass spectrometry : RCM", "id" : "ITEM-2", "issue" : "11", "issued" : { "date-parts" : [ [ "2011", "6", "15" ] ] }, "page" : "1559-1566", "title" : "Enhancements in travelling wave ion mobility resolution.", "type" : "article-journal", "volume" : "25" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "17,18", "plainTextFormattedCitation" : "17,18", "previouslyFormattedCitation" : "17,18" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }17,18 instruments (G1, G2 and newer) which can record the ATD for each ion. However, to obtain rotationally averaged collision cross sections, the TWIMS measurements need to be calibrated against samples with known CCS.ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/nprot.2008.78", "ISSN" : "1750-2799", "PMID" : "18600219", "abstract" : "Here we describe a detailed protocol for both data collection and interpretation with respect to ion mobility-mass spectrometry analysis of large protein assemblies. Ion mobility is a technique that can separate gaseous ions based on their size and shape. Specifically, within this protocol, we cover general approaches to data interpretation, methods of predicting whether specific model structures for a given protein assembly can be separated by ion mobility, and generalized strategies for data normalization and modeling. The protocol also covers basic instrument settings and best practices for both observation and detection of large noncovalent protein complexes by ion mobility-mass spectrometry.", "author" : [ { "dropping-particle" : "", "family" : "Ruotolo", "given" : "Brandon T", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Benesch", "given" : "Justin L P", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sandercock", "given" : "Alan M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hyung", "given" : "Suk-Joon", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "V", "family" : "Robinson", "given" : "Carol", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature protocols", "id" : "ITEM-1", "issue" : "7", "issued" : { "date-parts" : [ [ "2008", "1" ] ] }, "page" : "1139-1152", "title" : "Ion mobility-mass spectrometry analysis of large protein complexes.", "type" : "article-journal", "volume" : "3" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "19", "plainTextFormattedCitation" : "19", "previouslyFormattedCitation" : "19" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }19 Conversion of ATD to CCS is not strictly necessary, but it does remove the influence of ion charge, drift gas pressure, temperature, and other experimental variables thus making inter-lab comparisons more amenable. CCSs can be ‘learnt’ for any static structure using computational methods to mimic the experiment or data scaled from empirical values, and/or machine learning; here, also having experimental data in the form of CCS values facilitates comparison. For experiments which employ IM-MS to examine conformational changes in closely related molecular ions, for example apo and holo or mutated vs. wild type proteins, simply comparing IM-MS data taken on the same instrument can be highly informative.ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/ncomms12163", "ISBN" : "2041-1723 (Electronic)\\r2041-1723 (Linking)", "ISSN" : "2041-1723", "PMID" : "27418477", "abstract" : "Fdc1 is a decarboxylase enzyme that requires the novel prenylated FMN cofactor for activity. Here, we use it as an exemplar system to show how native top-down and bottom-up mass spectrometry can measure the structural effect of cofactor binding by a protein. For Fdc1Ubix, the cofactor confers structural stability to the enzyme. IM\u2013MS shows the holo protein to exist in four closely related conformational families, the populations of which differ in the apo form; the two smaller families are more populated in the presence of the cofactor and depopulated in its absence. These findings, supported by MD simulations, indicate a more open structure for the apo form. HDX-MS reveals that while the dominant structural changes occur proximal to the cofactor-binding site, rearrangements on cofactor binding are evident throughout the protein, predominantly attributable to allosteric conformational tightening, consistent with IM\u2013MS data.", "author" : [ { "dropping-particle" : "", "family" : "Beveridge", "given" : "Rebecca", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Migas", "given" : "Lukasz G.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Payne", "given" : "Karl A. P.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Scrutton", "given" : "Nigel S.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Leys", "given" : "David", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Barran", "given" : "Perdita E.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature Communications", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2016" ] ] }, "page" : "12163", "publisher" : "Nature Publishing Group", "title" : "Mass spectrometry locates local and allosteric conformational changes that occur on cofactor binding", "type" : "article-journal", "volume" : "7" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1002/pro.2699", "ISSN" : "1469896X", "PMID" : "25970849", "abstract" : "Cooperative binding mechanisms are a common feature in biology, enabling a diverse range of protein-based molecular machines to regulate activities ranging from oxygen uptake to cellular membrane transport. Much, however, is not known about such cooperative binding mechanisms, including how such events typically add to the overall stability of such protein systems. Measurements of such cooperative stabilization events are challenging, as they require the separation and resolution of individual protein complex bound states within a mixture of potential stoichiometries to individually assess protein stabilities. Here, we report ion mobility-mass spectrometry results for the concanavalin A tetramer bound to a range of polysaccharide ligands. We use collision induced unfolding, a relatively new methodology that functions as a gas-phase analog of calorimetry experiments in solution, to individually assess the stabilities of concanavalin A bound states. By comparing the differences in activation voltage required to unfold different concanavalin A\u2013ligand stoichiometries, we find evidence suggesting a cooperative stabilization of concanavalin A occurs upon binding most carbohydrate ligands. We critically evaluate this observation by assessing a broad range of ligands, evaluating the unfolding properties of multiple protein charge states, and by comparing our gas-phase results with those obtained from calorimetry experiments carried out in solution.", "author" : [ { "dropping-particle" : "", "family" : "Niu", "given" : "Shuai", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ruotolo", "given" : "Brandon T.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Protein Science", "id" : "ITEM-2", "issue" : "8", "issued" : { "date-parts" : [ [ "2015" ] ] }, "page" : "1272-1281", "title" : "Collisional unfolding of multiprotein complexes reveals cooperative stabilization upon ligand binding", "type" : "article-journal", "volume" : "24" }, "uris" : [ "" ] }, { "id" : "ITEM-3", "itemData" : { "DOI" : "10.1021/jacs.6b11678", "ISSN" : "0002-7863", "author" : [ { "dropping-particle" : "", "family" : "Eschweiler", "given" : "Joseph D.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Martini", "given" : "Rachel M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ruotolo", "given" : "Brandon T.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of the American Chemical Society", "id" : "ITEM-3", "issued" : { "date-parts" : [ [ "2016" ] ] }, "page" : "jacs.6b11678", "title" : "Chemical Probes and Engineered Constructs Reveal a Detailed Unfolding Mechanism for a Solvent-Free Multidomain Protein", "type" : "article-journal" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "4,20,21", "plainTextFormattedCitation" : "4,20,21", "previouslyFormattedCitation" : "4,20,21" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }4,20,21The process of gas phase protein restructuring monitored by mobility measurements, has recently been referred to as Collision Induced Unfolding (CIU), but perhaps more generally can be termed activated ion mobility mass spectrometry (aIM-MS). The reasons for introducing this term are manifold: in many collisional activation IM-MS experiments, proteins have been shown to first collapse before unfolding; it is also evident that proteins can refold upon collisional activation and most critically they can fragment, either by the loss of non-covalently bound subunits or cofactors, or by the breaking of covalent bonds. We also believe that this term may be useful for generally describing the use of IM-MS to monitor activation, perhaps by electrons with ETD or with photons in UVPD, or even with an IR laser where the term action spectroscopy is well known. An early demonstration of aIM-MS on proteins was conducted by Shelimov et al.ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "author" : [ { "dropping-particle" : "", "family" : "Shelimov", "given" : "Konstantin B", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Clemmer", "given" : "David E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hudgins", "given" : "Robert R", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Jarrold", "given" : "Martin F", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of the American Chemical Society", "id" : "ITEM-1", "issue" : "14", "issued" : { "date-parts" : [ [ "1997" ] ] }, "page" : "2240-2248", "title" : "Protein Structure in Vacuo : Gas-Phase Conformations of BPTI and Cytochrome c", "type" : "article-journal", "volume" : "7863" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "22", "plainTextFormattedCitation" : "22", "previouslyFormattedCitation" : "22" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }22 who investigated the stabilising influence of disulfide bonds by comparing the behaviour of BPTI (three disulfide bonds) with cytochrome c (none). Following these early experiments, aIM-MS was used in some analytical workflows but overall output remained low,ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "author" : [ { "dropping-particle" : "", "family" : "Clemmer", "given" : "David E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Jarrold", "given" : "Martin F", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of Mass Spectrometry", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "1997" ] ] }, "page" : "577-592", "title" : "Ion Mobility Measurements and their Applications to Clusters and Biomolecules", "type" : "article-journal", "volume" : "32" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "author" : [ { "dropping-particle" : "Von", "family" : "Helden", "given" : "Gert", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gotts", "given" : "Nigel", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bowers", "given" : "Michael T", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature", "id" : "ITEM-2", "issued" : { "date-parts" : [ [ "1993" ] ] }, "page" : "60-63", "title" : "Experimental evidence for the formation of fullerenes by collisional heating of carbon rings in the gas phase.", "type" : "article-journal", "volume" : "363" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "23,24", "plainTextFormattedCitation" : "23,24", "previouslyFormattedCitation" : "23,24" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }23,24 in line with a lack of commercial IM-MS instrumentation. Hopper et al. used a trap ion guide to kinetically energise protein-ligand complexes and be able to differentiate species based on their ATDs at elevated trap voltages.ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.jasms.2009.06.010", "ISSN" : "1879-1123", "PMID" : "19643633", "abstract" : "Ion mobility spectrometry, with subsequent mass spectrometric detection, has been employed to study the stability of compact protein conformations of FK-binding protein, hen egg-white lysozyme, and horse heart myoglobin in the presence and absence of bound ligands. Protein ions, generated by electrospray ionization from ammonium acetate buffer, were activated by collision with argon gas to induce unfolding of their compact structures. The collisional cross sections (Omega) of folded and unfolded conformations were measured in the T-Wave mobility cell of a Waters Synapt HDMS (Waters, Altrincham, UK) employing a calibration against literature values for a range of protein standards. In the absence of activation, collisional cross section measurements were found to be consistent with those predicted for folded protein structures. Under conditions of defined collisional activation energies partially unfolded conformations were produced. The degree of unfolding and dissociation induced by these defined collision energies are related to the stability of noncovalent intra- and intermolecular interactions within protein complexes. These findings highlight the additional conformational stability of protein ions in the gas phase resulting from ligand binding.", "author" : [ { "dropping-particle" : "", "family" : "Hopper", "given" : "Jonathan T S", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Oldham", "given" : "Neil J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of the American Society for Mass Spectrometry", "id" : "ITEM-1", "issue" : "10", "issued" : { "date-parts" : [ [ "2009", "10" ] ] }, "page" : "1851-8", "publisher" : "Elsevier Inc.", "title" : "Collision induced unfolding of protein ions in the gas phase studied by ion mobility-mass spectrometry: the effect of ligand binding on conformational stability.", "type" : "article-journal", "volume" : "20" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "14", "plainTextFormattedCitation" : "14", "previouslyFormattedCitation" : "14" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }14 More recently, aIM-MS methodology has been applied to more complicated systems to study the unfolding mechanisms of mono-, di- and tetrameric proteins,ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1002/anie.201403784", "ISBN" : "1521-3773 (Electronic)\\r1433-7851 (Linking)", "ISSN" : "1521-3773", "PMID" : "24990104", "abstract" : "The three-dimensional structures adopted by proteins are predicated by their many biological functions. Mass spectrometry has played a rapidly expanding role in protein structure discovery, enabling the generation of models for both proteins and their higher-order assemblies. While important coursed-grained insights have been generated, relatively few examples exist where mass spectrometry has been successfully applied to the characterization of protein tertiary structure. Here, we demonstrate that gas-phase unfolding can be used to determine the number of autonomously folded domains within monomeric proteins. Our ion mobility-mass spectrometry data highlight a strong, positive correlation between the number of protein unfolding transitions observed in the gas phase and the number of known domains within a group of sixteen proteins ranging from 8-78 kDa. This correlation and its potential uses for structural biology is discussed.", "author" : [ { "dropping-particle" : "", "family" : "Zhong", "given" : "Yueyang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Han", "given" : "Linjie", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ruotolo", "given" : "Brandon T.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Angewandte Chemie (International ed. in English)", "id" : "ITEM-1", "issue" : "35", "issued" : { "date-parts" : [ [ "2014" ] ] }, "page" : "1-5", "title" : "Collisional and Coulombic Unfolding of Gas-Phase Proteins: High Correlation to Their Domain Structures in Solution.", "type" : "article-journal", "volume" : "53" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1021/ac101121r", "ISBN" : "0003-2700", "ISSN" : "00032700", "PMID" : "20481443", "abstract" : "Tandem mass spectrometry (MS) of large protein complexes has proven to be capable of assessing the stoichiometry, connectivity, and structural details of multiprotein assemblies. While the utility of tandem MS is without question, a deeper understanding of the mechanism of protein complex dissociation will undoubtedly drive the technology into new areas of enhanced utility and information content. We present here the systematic analysis of the charge state dependent decay of the noncovalently associated complex of human transthyretin, generated by collision-induced dissociation (CID). A crown ether based charge reduction approach was applied to generate intact transthyretin tetramers with charge states ranging from 15+ to 7+. These nine charge states were subsequently analyzed by means of tandem MS and ion mobility spectrometry. Three different charge-dependent mechanistic regimes were identified: (1) common asymmetric dissociation involving ejection of unfolded monomers, (2) expulsion of folded monomers from the intact tetramer, and (3) release of C-terminal peptide fragments from the intact complex. Taken together, the results presented highlight the potential of charge state modulation as a method for directing the course of gas-phase dissociation and unfolding of protein complexes.", "author" : [ { "dropping-particle" : "", "family" : "Pagel", "given" : "Kevin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hyung", "given" : "Suk Joon", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ruotolo", "given" : "Brandon T.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "V.", "family" : "Robinson", "given" : "Carol", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Analytical Chemistry", "id" : "ITEM-2", "issue" : "12", "issued" : { "date-parts" : [ [ "2010" ] ] }, "page" : "5363-5372", "title" : "Alternate dissociation pathways identified in charge-reduced protein complex ions", "type" : "article-journal", "volume" : "82" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "25,26", "plainTextFormattedCitation" : "25,26", "previouslyFormattedCitation" : "25,26" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }25,26 differentiate biosimilarsADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/acs.analchem.5b03291", "ISSN" : "0003-2700", "author" : [ { "dropping-particle" : "", "family" : "Tian", "given" : "Yuwei", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Han", "given" : "Linjie", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Buckner", "given" : "Adam C.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ruotolo", "given" : "Brandon T.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Analytical Chemistry", "id" : "ITEM-1", "issue" : "22", "issued" : { "date-parts" : [ [ "2015" ] ] }, "page" : "11509-11515", "title" : "Collision Induced Unfolding of Intact Antibodies: Rapid Characterization of Disulfide Bonding Patterns, Glycosylation, and Structures", "type" : "article-journal", "volume" : "87" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1016/j.jchromb.2016.03.044", "ISBN" : "1873-376X (Electronic)\r1570-0232 (Linking)", "ISSN" : "1873376X", "PMID" : "27108304", "abstract" : "Over the past 15 years, monoclonal antibodies (mAbs) have emerged as the most successful class of therapeutics. Their specific structural and functional properties make them highly effective treatments for various diseases. Most therapeutic mAbs are based on chimeric, humanized or human G immunoglobulins (IgGs) selected from three isotypes (1, 2 and 4). IgGs are large and highly complex multimeric glycoproteins. They are constituted of a mixture of isoforms including macro and micro-variants that must be extensively characterized prior to their investigation as a drug candidate in clinical trials. The IgG backbone is also used to design more potent but also more complex biopharmaceuticals such as antibody-drug conjugates, bispecific antibodies, Fc-fusion proteins, and antibody mixtures to name a few. Mass spectrometric approaches in combination with electrophoretic and chromatographic separation methods play a central role in the analytical and structural multi-level (top, middle and bottom) characterization of these compounds. Importantly, techniques allowing the characterization of intact mAbs and related products under non-denaturing conditions are attracting increasing interest. Here, we review the current state of the art in native mass spectrometry and ion mobility methods for the characterization of mAbs and mAb-based products.", "author" : [ { "dropping-particle" : "", "family" : "Terral", "given" : "Guillaume", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Beck", "given" : "Alain", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Cianf??rani", "given" : "Sarah", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences", "id" : "ITEM-2", "issued" : { "date-parts" : [ [ "2016" ] ] }, "page" : "79-90", "publisher" : "Elsevier B.V.", "title" : "Insights from native mass spectrometry and ion mobility-mass spectrometry for antibody and antibody-based product characterization", "type" : "article-journal", "volume" : "1032" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "27,28", "plainTextFormattedCitation" : "27,28", "previouslyFormattedCitation" : "27,28" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }27,28 and observe alterations to protein stability upon single point mutationADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/jacs.6b11678", "ISSN" : "0002-7863", "author" : [ { "dropping-particle" : "", "family" : "Eschweiler", "given" : "Joseph D.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Martini", "given" : "Rachel M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ruotolo", "given" : "Brandon T.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of the American Chemical Society", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2016" ] ] }, "page" : "jacs.6b11678", "title" : "Chemical Probes and Engineered Constructs Reveal a Detailed Unfolding Mechanism for a Solvent-Free Multidomain Protein", "type" : "article-journal" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "21", "plainTextFormattedCitation" : "21", "previouslyFormattedCitation" : "21" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }21 and ligand/cofactor binding.ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1007/s13361-016-1496-8", "ISSN" : "1044-0305", "abstract" : "Fibroblast growth factors (FGFs) regulate several cellular developmental processes by interacting with cell surface heparan proteoglycans and transmembrane\\r\\ncell surface receptors (FGFR). The interaction of FGF with heparan sulfate (HS) is known to induce protein oligomerization, increase the affinity of FGF towards its receptor FGFR, promoting the formation of the HS\u2013FGF\u2013FGFR signaling complex. Although the role of\\r\\nHS in the signaling pathways is well recognized, the details of FGF oligomerization and formation of the ternary signaling complex are still not clear, with several conflicting models proposed in literature. Here, we examine the effect of size and sulfation pattern\\r\\nof HS upon FGF1 oligomerization, binding stoichiometry and conformational stability, through a combination of ion mobility (IM) and theoretical modeling approaches. Ion mobility-mass spectrometry (IMMS) of FGF1 in the presence of several HS fragments ranging from tetrasaccharide (dp4) to dodecasaccharide (dp12) in length was performed. A comparison of the binding stoichiometry of variably sulfated dp4 HS to FGF1 confirmed the significance of the previously known high-affinity binding motif in FGF1 dimerization, and demonstrated that certain tetrasaccharide-length fragments are also capable of inducing dimerization\\r\\nof FGF1. The degree of oligomerization was found to increase in the presence of dp12 HS, and a general lack of specificity for longer HS was observed. Additionally, collision cross-sections (CCSs) of several FGF1\u2013HS complexes were calculated, and were found to be in close agreement with experimental results. Based on the (CCSs) a number of plausible binding modes of 2:1 and 3:1 FGF1\u2013HS are proposed.", "author" : [ { "dropping-particle" : "", "family" : "Zhao", "given" : "Yueije", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Singh", "given" : "Arunima", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Xu", "given" : "Yongmei", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Zong", "given" : "Chengli", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Zhang", "given" : "Fuming", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Boons", "given" : "Geert-Jan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Liu", "given" : "Jian", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Linhardt", "given" : "j Robert J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Woods", "given" : "Robert J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Amster", "given" : "Jonathan I", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of the American Society for Mass S", "id" : "ITEM-1", "issue" : "335", "issued" : { "date-parts" : [ [ "2016" ] ] }, "page" : "1-14", "title" : "Gas-Phase Analysis of the Complex of Fibroblast Growth Factor 1 with Heparan Sulfate: A Traveling Wave Ion Mobility Spectrometry (TWIMS) and Molecular Modeling Study", "type" : "article-journal", "volume" : "27" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1002/pro.2699", "ISSN" : "1469896X", "PMID" : "25970849", "abstract" : "Cooperative binding mechanisms are a common feature in biology, enabling a diverse range of protein-based molecular machines to regulate activities ranging from oxygen uptake to cellular membrane transport. Much, however, is not known about such cooperative binding mechanisms, including how such events typically add to the overall stability of such protein systems. Measurements of such cooperative stabilization events are challenging, as they require the separation and resolution of individual protein complex bound states within a mixture of potential stoichiometries to individually assess protein stabilities. Here, we report ion mobility-mass spectrometry results for the concanavalin A tetramer bound to a range of polysaccharide ligands. We use collision induced unfolding, a relatively new methodology that functions as a gas-phase analog of calorimetry experiments in solution, to individually assess the stabilities of concanavalin A bound states. By comparing the differences in activation voltage required to unfold different concanavalin A\u2013ligand stoichiometries, we find evidence suggesting a cooperative stabilization of concanavalin A occurs upon binding most carbohydrate ligands. We critically evaluate this observation by assessing a broad range of ligands, evaluating the unfolding properties of multiple protein charge states, and by comparing our gas-phase results with those obtained from calorimetry experiments carried out in solution.", "author" : [ { "dropping-particle" : "", "family" : "Niu", "given" : "Shuai", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ruotolo", "given" : "Brandon T.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Protein Science", "id" : "ITEM-2", "issue" : "8", "issued" : { "date-parts" : [ [ "2015" ] ] }, "page" : "1272-1281", "title" : "Collisional unfolding of multiprotein complexes reveals cooperative stabilization upon ligand binding", "type" : "article-journal", "volume" : "24" }, "uris" : [ "" ] }, { "id" : "ITEM-3", "itemData" : { "DOI" : "10.1038/ncomms12163", "ISBN" : "2041-1723 (Electronic)\\r2041-1723 (Linking)", "ISSN" : "2041-1723", "PMID" : "27418477", "abstract" : "Fdc1 is a decarboxylase enzyme that requires the novel prenylated FMN cofactor for activity. Here, we use it as an exemplar system to show how native top-down and bottom-up mass spectrometry can measure the structural effect of cofactor binding by a protein. For Fdc1Ubix, the cofactor confers structural stability to the enzyme. IM\u2013MS shows the holo protein to exist in four closely related conformational families, the populations of which differ in the apo form; the two smaller families are more populated in the presence of the cofactor and depopulated in its absence. These findings, supported by MD simulations, indicate a more open structure for the apo form. HDX-MS reveals that while the dominant structural changes occur proximal to the cofactor-binding site, rearrangements on cofactor binding are evident throughout the protein, predominantly attributable to allosteric conformational tightening, consistent with IM\u2013MS data.", "author" : [ { "dropping-particle" : "", "family" : "Beveridge", "given" : "Rebecca", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Migas", "given" : "Lukasz G.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Payne", "given" : "Karl A. P.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Scrutton", "given" : "Nigel S.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Leys", "given" : "David", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Barran", "given" : "Perdita E.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature Communications", "id" : "ITEM-3", "issued" : { "date-parts" : [ [ "2016" ] ] }, "page" : "12163", "publisher" : "Nature Publishing Group", "title" : "Mass spectrometry locates local and allosteric conformational changes that occur on cofactor binding", "type" : "article-journal", "volume" : "7" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "4,20,29", "plainTextFormattedCitation" : "4,20,29", "previouslyFormattedCitation" : "4,20,29" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }4,20,29 aIM-MS in a Synapt ion mobility-mass spectrometer is facilitated by sequentially increasing the potential difference between the source and trap region of the instrument which effectively raises the ions kinetic energy in the presence of the trapping gas, followed by IM-MS separation which can then be used to monitor the influence of activation on the ion of interest. Unsurprisingly, as an ions kinetic energy increases, its conformation can adapt in order to accommodate its excessive internal energy prior to fragmentation. This process often leads to a marked change in the recorded ATD. Despite IM-MS being a relatively low resolution technique in comparison to other biophysical methods (i.e. X-ray crystallography or NMR), it offers multiple benefits including low sample consumption, relatively quick acquisition and analysis times and the potential for high throughput screening. In spite of these benefits, when applied to large macromolecular complexes such as proteins, aIM-MS methodology contains several bottlenecks that preclude its wide adoption. The current barriers include: (i) sequential ramping of collision voltages (CV) requires either generation of a sample list that would automate acquisition or manually acquire file for each CV; (ii) current protocols result in multiple files, and while it is possible to perform CV ramp within a single file, the user has little control over the experimental parameters (i.e. step size); (iii) long lists of files lead to large volumes of data handling; (iv) extraction of ATDs for an ion at given CVs is time consuming, not systematic and easily varies from user to user. While there are existing open-source software packages capable of efficient data extraction (PULSARADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/ncomms9551", "ISBN" : "2041-1723 (Electronic)\r2041-1723 (Linking)", "ISSN" : "2041-1723", "PMID" : "26440106", "abstract" : "The effects of protein-ligand interactions on protein stability are typically monitored by a number of established solution-phase assays. Few translate readily to membrane proteins. We have developed an ion-mobility mass spectrometry approach, which discerns ligand binding to both soluble and membrane proteins directly via both changes in mass and ion mobility, and assesses the effects of these interactions on protein stability through measuring resistance to unfolding. Protein unfolding is induced through collisional activation, which causes changes in protein structure and consequently gas-phase mobility. This enables detailed characterization of the ligand-binding effects on the protein with unprecedented sensitivity. Here we describe the method and software required to extract from ion mobility data the parameters that enable a quantitative analysis of individual binding events. This methodology holds great promise for investigating biologically significant interactions between membrane proteins and both drugs and lipids that are recalcitrant to characterization by other means.", "author" : [ { "dropping-particle" : "", "family" : "Allison", "given" : "Timothy M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Reading", "given" : "Eamonn", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Liko", "given" : "Idlir", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Baldwin", "given" : "Andrew J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Laganowsky", "given" : "Arthur", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "V", "family" : "Robinson", "given" : "Carol", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature communications", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2015" ] ] }, "page" : "8551", "publisher" : "Nature Publishing Group", "title" : "Quantifying the stabilizing effects of protein-ligand interactions in the gas phase.", "type" : "article-journal", "volume" : "6" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "30", "plainTextFormattedCitation" : "30", "previouslyFormattedCitation" : "30" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }30 and TWIMExtractADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/acs.analchem.7b00112", "author" : [ { "dropping-particle" : "", "family" : "Haynes", "given" : "Sarah E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Polasky", "given" : "Daniel A", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Dixit", "given" : "Sugyan M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Majmudar", "given" : "Jaimeen D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Neeson", "given" : "Kieran", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ruotolo", "given" : "Brandon T", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Martin", "given" : "Brent R", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2017" ] ] }, "page" : "4-7", "title" : "Variable-Velocity Traveling-Wave Ion Mobility Separation Enhancing Peak Capacity for Data-Independent Acquisition Proteomics", "type" : "article-journal" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "31", "plainTextFormattedCitation" : "31", "previouslyFormattedCitation" : "31" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }31), sophisticated data analysis (UniDec)ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/acs.analchem.5b00140", "ISBN" : "0003-2700", "ISSN" : "15206882", "PMID" : "23850452", "abstract" : "We report the design and first applications of a tandem mass spectrometer (a quadrupole time-of-flight mass spectrometer) optimized for the transmission and analysis of large macromolecular assemblies. Careful control of the pressure gradient in the different pumping stages of the instrument has been found to be essential for the detection of macromolecular particles. Such assemblies are, however, difficult to analyze by tandem-MS approaches, because they give rise to signals above m/z 3,000-4,000, the limit for commercial quadrupoles. By reducing the frequency of the quadrupole to 300 kHz and using it as a narrow-band mass filter, we show that it is possible to isolate ions from a single peak at m/z 22,000 in a window as narrow as 22 m/z units. Using cesium iodide cluster signals, we show that the mass range in the time-of-flight (TOF) analyzer extends beyond m/z 90,000, in theory to more than m/z 150,000. We also demonstrate that the resolution of the instrument is greater than 3,000 at m/z 44,500. Tandem-MS capabilities are illustrated by separating components from heterooligomeric assemblies formed between tetrameric transthyretin, thyroxine, retinol-binding protein, and retinol. Isolation of a single charge state at m/z 5,340 in the quadrupole and subsequent collision-induced dissociation (CID) in the gas-filled collision cell leads to the formation of ions from individual subunits and subcomplexes, identified by their mass and charge in the TOF analyzer.", "author" : [ { "dropping-particle" : "", "family" : "Marty", "given" : "Michael T.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Baldwin", "given" : "Andrew J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Marklund", "given" : "Erik G.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hochberg", "given" : "Georg K A", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Benesch", "given" : "Justin L P", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "V.", "family" : "Robinson", "given" : "Carol", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Analytical Chemistry", "id" : "ITEM-1", "issue" : "8", "issued" : { "date-parts" : [ [ "2015" ] ] }, "page" : "4370-4376", "title" : "Bayesian deconvolution of mass and ion mobility spectra: From binary interactions to polydisperse ensembles", "type" : "article-journal", "volume" : "87" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "32", "plainTextFormattedCitation" : "32", "previouslyFormattedCitation" : "32" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }32 and data visualisation (CIUSuite)ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/acs.analchem.5b03292", "ISSN" : "0003-2700", "author" : [ { "dropping-particle" : "", "family" : "Eschweiler", "given" : "Joseph D.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rabuck-Gibbons", "given" : "Jessica N.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tian", "given" : "Yuwei", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ruotolo", "given" : "Brandon T.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Analytical Chemistry", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2015" ] ] }, "page" : "acs.analchem.5b03292", "title" : "CIUSuite: A Quantitative Analysis Package for Collision Induced Unfolding Measurements of Gas-Phase Protein Ions", "type" : "article-journal" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "33", "plainTextFormattedCitation" : "33", "previouslyFormattedCitation" : "33" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }33, ORIGAMI offers a full suite of tools for efficient aIM acquisition, high-throughput analysis and detailed data visualisation in one place. In this report we present a novel, open-source software package for semi-autonomous activated IM-MS data acquisition (ORIGAMIMS) for Synapt G2, G2S and G2Si instruments, with our complementary rapid data analysis tool (ORIGAMIANALYSE). (ORIGAMIMS) automates the aIM process by sequentially raising the collision voltage within a single MassLynx file, whilst giving users full control over the activation parameters. ORIGAMIANALYSE enables direct interrogation of ORIGAMIMS and manually (or sample list) acquired datasets, thus simplifying the data processing but more importantly reducing the quantity of data handling and analysis time. ORIGAMIMS reduces the experimental footprint (number and size of files) and improves the reproducibility and reliability of aIM-MS protocols. In order to demonstrate some of the capabilities and benefits of using ORIGAMI, we present its use on two multimeric proteins that are readily available as standard molecules. Materials and methodsMaterialsConcanavalin A (ConA, jack bean), alcohol dehydrogenase (AdH, Saccharomyces cerevisiae), and ammonium acetate were purchased from Sigma (St. Louis, MO, USA). All protein samples were buffer exchanged into ammonium acetate at pH 7.4 using Micro Bio-Spin 6 columns (Bio-Rad, Hercules, CA, USA) and prepared to a final concentration of 10 μM (ConA, AdH; 200mM AmAc). Either fresh or flash frozen samples thawed on ice were used for analysis.Experimental conditionsAll experiments were carried out on an unmodified Synapt G2 and G2S (Waters, Wilmslow, UK) in positive ionisation mode with nitrogen as the buffer gas. Samples were ionized by applying a positive potential through a platinum wire (Diameter 0.125 mm, Goodfellow, Huntingdon, UK) inserted into nESI tips that were pulled in-house from thin-walled glass capillaries (i.d. 0.69 mm, o.d. 1.2 mm, Sutter Instrument Company, Novato, CA, USA.) using a P2000/F laser puller (Sutter Instrument Co., Novato, CA, USA). All ions were generated via electrospray ionisation using a capillary voltage of 0.7-1.2 kV, a sampling cone of 10-20 V and a source temperature of 40 °C. All other experimental voltages (including travelling wave settings) were minimised in order to reduce protein activation prior to the collision voltage ramping process. Detailed parameters are shown in SI Table 1. In order to raise the transmission efficiency of ConA and AdH, the backing pressure was set to 5-7.5x101 mbar and the Trap cell gas flow to 4-7 mL/min. Collisional activation of analytes was accomplished by sequentially increasing the collision voltage in the Trap collision cell prior to ion mobility separation using both ORIGAMIMS and the manual protocols. The collision voltage was typically ramped from 4 to 200 V in 2 V increments. Detailed parameters of the aIM-MS process are shown in SI Table 2. An exemplar charge state ([AdH+24H]24+ and [ConA+20H]20+) for each protein was first mass selected in the quadrupole prior to activated ion mobility separation; mass spectra and arrival time distributions were collected in MSMS mode.Software designORIGAMI is an open-source software package consisting of two components, ORIGAMIMS, capable of streamlined aIM-MS data acquisition and ORIGAMIANALYSE, used to extract, analyse and plot activation fingerprint data. The software was developed using Python 2.7 programming language utilising several popular modules, namely Numpy,ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1109/MCSE.2007.58", "ISBN" : "1521-9615", "ISSN" : "1521-9615", "author" : [ { "dropping-particle" : "", "family" : "Lima", "given" : "Ivan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Marine Chemistry", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2006" ] ] }, "page" : "10-20", "title" : "Python for Scientific Computing Python Overview", "type" : "article-journal" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "34", "plainTextFormattedCitation" : "34", "previouslyFormattedCitation" : "34" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }34 SciPy, MatplotlibADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1109/MCSE.2007.55", "ISBN" : "1521-9615 VO - 9", "ISSN" : "15219615", "PMID" : "1000044628", "abstract" : "Matplotlib is a 2D graphics package used for Python for application development, interactive scripting, and publication-quality image generation across user interfaces and operating systems.", "author" : [ { "dropping-particle" : "", "family" : "Hunter", "given" : "John D.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Computing in Science and Engineering", "id" : "ITEM-1", "issue" : "3", "issued" : { "date-parts" : [ [ "2007" ] ] }, "page" : "99-104", "title" : "Matplotlib: A 2D graphics environment", "type" : "article-journal", "volume" : "9" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "35", "plainTextFormattedCitation" : "35", "previouslyFormattedCitation" : "35" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }35 and Bokeh amongst many others. A graphical user interface (GUI) was developed in wxPython. Both programs are distributed as pre-compiled executables available from , free of charge for academic use. The source code of both components of ORIGAMI can be obtained from autonomous ORIGAMIMS software was developed in C# using the Waters Research Enabled Software (WREnS) which enables additional controls of the Synapt hardware. In order to achieve controlled collisional activation independently of MassLynx, WREnS is used to control ion transmission by modulation of DC potentials, in particular for the DRE lens which controls ion signal attenuation and ion source and trap collision cell voltages. While no hardware modifications are required to utilise ORIGAMIMS, the user is required to install WREnS on their instrument PCs. WREnS can be obtained by research agreement from Waters (Wilmslow, UK) with installation instructions. ORIGAMIMS can be used in three ways, utilising the user-friendly GUI (shown in Figure S1), executed from the command line or as a pre-process program in the MassLynx worklist. While integration of custom WREnS scripts into acquisition protocols can be beneficial, it is not necessary. The users can automate the acquisition of aIM datasets by integration of MassLynx ‘sample lists’ which possess the experimental conditions for each individual ‘sample’ (i.e. ramped collision voltage).The analysis software, ORIGAMIANALYSE accepts several data types, including MassLynx .raw files, text files and correctly formatted Microsoft Excel spreadsheets. Rather than ‘reinventing the wheel’ we have adopted components of the Driftscope software (Waters, Wilmslow, UK) to read MassLynx .raw files, supplied with the Synapt hardware. Driftscope enables quick extraction of the IM-MS data in a binary format which can be readily converted to a [n x 200] matrix, where n refers to the number of scans, which in turn may track a collision voltage ramp or indeed any other activation process that might subsequently be monitored by IM-MS (Surface Induced Dissociation, SID; Infrared Multiphoton Dissociation, IRMPD; and others). The program has no specific pre-requisites, however in order to extract MS and IM-MS data from MassLynx .raw files, a local installation of Driftscope is required. Presently, data extraction is only available on Windows platforms as for all other analyses of MassLynx data. The analysis software was built and tested on Microsoft Windows 7/8/10 with 64-bit hardware architecture. ORIGAMIANALYSE can import either single or multiple MassLynx files simultaneously, providing the same analysis support for both manually and autonomously acquired datasets. Regardless of the input format, mass spectra are loaded as a sum of all scans for each loaded MassLynx file. In the case of ORIGAMIMS acquired data, the default displayed mass spectrum is a composite of all scans within the file consisting of all collision voltage increments; individual mass spectra for specific collision voltages can be extracted and saved separately in a text format. Initially, the 2D/3D aIM plot is shown for the entire mass spectrum, typically consisting of a combination of precursor and fragment ions. In order to examine single charge states, the user can select a m/z region of interest permitting the corresponding ATD profile for each collision voltage to be extracted and stitched together to form an aIM matrix. ORIGAMIANALYSE is capable of mining multiple ions simultaneously and saving them in a text or compressed binary format for storage or further analysis in other software packages. Figure S2 shows the graphical user interface used to analyse ORIGAMIMS acquired datasets, the multi-ion selection tool is highlighted, as well as the interface shown during analysis of multiple text files and processing of lists of MassLynx .raw files ResultsThe ORIGAMIMS workflowThe backbone of the ORIGAMI package is ORIGAMIMS, which has been designed as a user-friendly program to develop advanced mass spectrometry workflows. Here it is used to automate an increase in applied collision voltages to induce ion restructuring and/or dissociation. On Waters Synapt instruments, collisional activation can occur prior to mass selection in the sampling cone, post the quadrupole in the trap collision cell, or post TWIM separation in the transfer collision cell, on either mass selected or all molecular ions. Here we only explore activation in the trap collision cell, but we have also implemented a mode for sampling cone activation, which is more suited to analysis where the analyte presents in a single charge state. The collision voltage is slowly raised from CESTART to CEEND with a small increment CESTEP. In ORIGAMIMS, the user can specify the region of the mass spectrometer where collisional activation is to be performed (i.e. cone or trap), ion polarity, starting and ending voltage, step size, scan time and time spent on each collision voltage. Once an acquisition is started, the collision voltage ramp is performed autonomously and the data is recorded to a single MassLynx file. Figure 1. Procedure for activated IM-MS with a collision voltage ramp carried out using ORIGAMIMS. (a) Schematic to show the sequence of a typical experiment wherein the collision voltage is raised from 5 V to 35V with a 5 V increment. In this example, data is acquired for three scans at each collision voltage with a pre-set scan time of 5 s (15 s per 5 V, shown in inset). The area highlighted in green represents the initial experimental conditions (before and after the aIM-MS experiment) and the blue area shows the two periods for which the ion signal is attenuated to < 1 % of its original value in order to delineate when the aIM-MS process has started and finished. The entire experiment in this example takes ~2.5 min. (b) a reference table which shows the approximate acquisition times based on a pre-set scan time of 5 s, with a CE increment of 2 V and 3 scans per voltage. (c) Typical experimental parameters and the minimum/maximum ranges.A simplified example of the ORIGAMIMS workflow is shown in Figure 1a. In this exemplar case, the area shown in red depicts the collision voltage profile as the voltage is raised. Here the collision voltage is raised from 5 to 35 V with an increment of 5 V. Each collision voltage is kept constant for 3 scans with a scan time of 5 seconds, resulting in 15 seconds of acquisition per voltage (Figure 1a inset). The two areas shown in blue (before and after the CE ramp) represent ‘reporter’ periods in which the ion flow is stopped in order for ORIGAMIANALYSE to determine when the activation process has started and stopped. The areas shown in green represent the initial experimental conditions which are reset at the end of the aIM-MS protocol. The procedure shown in Figure 1a gives a total data acquisition time of 2.5 minutes, however in most experiments the voltage ramp will span a wider range of values. The approximate acquisition times shown in Figure 1b are for experiments using voltage increments of 2 V with 3 scans per voltage and a scan time of 5 seconds. Typical experimental parameters and their ranges are shown in Figure 1c. Often as a consequence of increasing the collision voltage, the signal intensity of the precursor ion decays rapidly. In order to account for the decrease in the ion intensity we have developed three methods where the number of scans per voltage (SPV) increase as a function of the collision voltage. The original method is referred to as linear (shown above), whilst the more advanced methods include exponential, Boltzmann and user-specified; these methods have been developed via extensive aIM-MS studies encompassing a range of biomolecules (data not shown). Briefly, the exponential method increases the number of SPV as a product of the exponential function scaled by two user-specified parameters which define the relative starting point and rate of the SPV rise. The Boltzmann method is based on a modified Boltzmann equation fitted to multiple datasets that represent the common ion intensity decay rate amongst multiple biomolecules. The Boltzmann method accepts one user-specified parameter which defines the rate of SPV increase. Finally, the user-specified method accepts a list of SPVs for each collision voltage supplied by the user. These non-linear ramp functions can improve the signal-to-noise for the precursor and fragment ions but also lead to longer acquisition times. A comparison of the three methods (linear, exponential and Boltzmann) is shown in Figure S3 where the relative number of SPV (a), acquisition time (b) and extracted ion current (c) of the precursor ion ([4M+20H]20+ ConA) is presented. The collision activation range was set to 4-200 V with an increment of 2 V, initial 3 scans per voltage and scan time of 5 s. In the case of the exponential method, the value of SPV was set to start rising from 60 V at a rate of 0.02 whilst the Boltzmann method used an offset of 55. Figure S3a shows the rise in the number of SPV, where the value remained constant for the linear method, but increased to 12 and 15 for the exponential and Boltzmann methods respectively. In addition, the total acquisition time nearly doubled from 25.5 to 47 min for the exponential and to 55 mins for the Boltzmann method. As a consequence of the increase in SPV, the rate of decay of the extracted ion current (EIC) was reduced, especially for the Boltzmann method, where the EIC is significantly more intense in comparison to the linear method (at 92, 134 and 164 V). Similar observation can be made for the exponential method, however the increase in EIC at particular voltages is less pronounced. The improved signal-to-noise ratio of the precursor ion as a function of collision voltage results in increased resolution of the 2D aIM-MS plots which are shown in Figure S4 and in the Supplementary File 2. ORIGAMIANALYSE overviewORIGAMIANALYSE was developed to tackle one of the major bottlenecks of the activated IM-MS protocol, namely the time-consuming data processing, as well as to enhance data visualisation. Our aims were: to create software capable of reading MassLynx and text files; to accelerate data processing; and to allow the user to better view the acquired data which in turn will assist interpretation of the experiment performed. A summary of functions and plots available in the program is shown in Figure 2, some of which are discussed in more detail below and in the SI. The majority of built-in methods can operate on MassLynx, text and Excel files, with the obvious exception of m/z data extraction. A typical workflow of acquisition and analysis of ORIGAMIMS with ORIGAMIANALYSE is shown in Figure S5, in this case data is shown for the alcohol dehydrogenase 24+ ion. The software can be operated in two ways, using the GUI or in a high-throughput batch mode using the command line. 2D and 3D visualisation of aIM datasets are the most informative way of looking at activated analyte fingerprints. These plots convey information about the ATD profiles at individual collision voltages, adequately display the relative intensity of conformational families and easily highlight trends and unfolding motifs. The visualisation window offers easy navigation and a high level of interaction, enabling detailed analysis of specific regions of the aIM fingerprints. Additionally, all plots made in ORIGAMIANALYSE can be exported in a .html format, producing fully interactive and shareable documents (i.e. could be used as Supplementary Files). An example of one is attached as Supplementary File 2, in this case it shows the comparison of 2D aIM-MS plots from the linear, exponential and Boltzmann methods of [4M+20H]20+ ConA (same as Figure S4). The raw data can be normalised, smoothed, interpolated and filtered within ORIGAMIANALYSE in order to improve the image quality. Figure 2. General list of features available in ORIGAMIANALYSE. A more detailed description of the available plots is included in the SI.aIM Fingerprint comparisonA common visualisation technique to observe structural, conformational and stability differences between samples is to use a subtraction heatmap and root mean square deviation (RMSD) calculation, as previously shown in CIUSuite.ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/acs.analchem.5b03292", "ISSN" : "0003-2700", "author" : [ { "dropping-particle" : "", "family" : "Eschweiler", "given" : "Joseph D.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rabuck-Gibbons", "given" : "Jessica N.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tian", "given" : "Yuwei", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ruotolo", "given" : "Brandon T.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Analytical Chemistry", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2015" ] ] }, "page" : "acs.analchem.5b03292", "title" : "CIUSuite: A Quantitative Analysis Package for Collision Induced Unfolding Measurements of Gas-Phase Protein Ions", "type" : "article-journal" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "33", "plainTextFormattedCitation" : "33" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }33 These can be used to quantify the difference between datasets by subtracting two aIM matrices and computing the pairwise difference. The RMSD can be calculated using equation 1:RMSD= A-B2m × n ×100 %(1)where A and B are aIM data matrices of identical size with the dimensions of [n x m] (n is the number of collision voltage points and m is the number of IM-MS bins). Another way to visualise the RMSD contour map is to plot the RMSD as a function of the collision voltage (RMSDCV), calculated using equation 2: RMSDCVn= An-Bn2m ×100 %(2)where A and B are aIM lists for individual n with the dimensions of m. RMSD plots (Figure 3b) are more sensitive to ‘global’ differences in the collisional unfolding of analytes and can easily discriminate between permutants, single and multi-point mutations or the effects of ligand binding on protein stability. In contrast, the RMSDCV (Figure 3a) offers a ‘local’ insight into the motifs as it examines the minor fluctuations occurring at each collision voltage. The RMSDCV can help identify differences at specific collision voltages and is most suited for day-to-day and batch-to-batch comparisons in addition to applications mentioned above. Figure 3. Comparison of manually and ORIGAMIMS acquired datasets spanning identical collision voltage ranges for the ConA [4M+20H]20+ ion. (a) RMSDCV plot highlighting collision voltages with most significant differences in their arrival time distributions and relative intensities. (b) A subtraction plot of two matrices with an overall RMSD error of 4.61 %. (c) An overlay plot of the two datasets with manually acquired data shown in red and ORIGAMIMS in green. The plots were generated by applying a mask of different colour to each dataset individually. While the usage of RMSD values and subtraction plots is unequivocally useful, the technique is reliant on the two datasets being of identical size and encompassing the same collision voltage range. In the case where n or the individual collision voltages differ, the subtraction plot will be misaligned and the corresponding RMSD values greatly exaggerated; minor shifts in the ATD profile which can be caused by a number of external factors (i.e. differences in IMS or trap pressures, source conditions, or intermittent spray) can lead to over-stated RMSD differences. In order to alleviate some of these issues, the aIM data matrices are typically averaged over three replicates obtained on separate days, allowing the average aIM data fingerprints to be compared. In most cases the ion of interest occupies a fraction of the aIM data, hence the matrix can be cropped or a mask (threshold) applied to remove noise peaks, this however tends to increase the RMSD values. Typical image processing methods, such as image smoothing, noise suppression and image interpolation can be used and have significant impact on the RMSD score and the subtraction plot. Another method to visualize the aIM fingerprints is to overlay two (or more) matrices and represent them as individually coloured masks (shown in Figure 3c). While the overlay method provides no statistical or numerical information about the difference (or similarity) of the two analytes, it enables direct comparison of multiple matrices and offers clear recognition of regions of interest (also shown in Figure 4c). aIM fragment overlayInformation obtained from a single activated IM-MS experiment can complement non activated IM-MS and non IM tandem mass spectrometry experiments by providing detailed information about ion unfolding pathways, stabilities and conformational diversity. With the implementation of ORIGAMIMS into the analytical workflow, the collision voltage ramp can be fully controlled and accomplished with speed and ease. Unsurprisingly, during collisional activation the kinetically excited precursor ion can undergo CID fragmentation, which is dependent on multiple parameters including inter- and intramolecular bonds, applied collision voltage and ion charge state.ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/ac101121r", "ISBN" : "0003-2700", "ISSN" : "00032700", "PMID" : "20481443", "abstract" : "Tandem mass spectrometry (MS) of large protein complexes has proven to be capable of assessing the stoichiometry, connectivity, and structural details of multiprotein assemblies. While the utility of tandem MS is without question, a deeper understanding of the mechanism of protein complex dissociation will undoubtedly drive the technology into new areas of enhanced utility and information content. We present here the systematic analysis of the charge state dependent decay of the noncovalently associated complex of human transthyretin, generated by collision-induced dissociation (CID). A crown ether based charge reduction approach was applied to generate intact transthyretin tetramers with charge states ranging from 15+ to 7+. These nine charge states were subsequently analyzed by means of tandem MS and ion mobility spectrometry. Three different charge-dependent mechanistic regimes were identified: (1) common asymmetric dissociation involving ejection of unfolded monomers, (2) expulsion of folded monomers from the intact tetramer, and (3) release of C-terminal peptide fragments from the intact complex. Taken together, the results presented highlight the potential of charge state modulation as a method for directing the course of gas-phase dissociation and unfolding of protein complexes.", "author" : [ { "dropping-particle" : "", "family" : "Pagel", "given" : "Kevin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hyung", "given" : "Suk Joon", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ruotolo", "given" : "Brandon T.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "V.", "family" : "Robinson", "given" : "Carol", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Analytical Chemistry", "id" : "ITEM-1", "issue" : "12", "issued" : { "date-parts" : [ [ "2010" ] ] }, "page" : "5363-5372", "title" : "Alternate dissociation pathways identified in charge-reduced protein complex ions", "type" : "article-journal", "volume" : "82" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "26", "plainTextFormattedCitation" : "26", "previouslyFormattedCitation" : "26" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }26 For large macromolecular complexes collisional activation can cause unfolding, and fragmentation patterns may be a readout of structural rearrangements. Whilst it is common in tandem mass spectrometry to present the fragment ions, to date, aIM-MS work has principally considered the effects of activation on the precursor ion with an experimental workflow that normalises the data from each collision voltage. Since this latter approach does not present all of the information from the experiment, for instance the unfolding pathways of fragment ions, we have developed ORIGAMIANALYSE to address this. ORIGAMIANALYSE allows direct, facile extraction of multiple ions from MassLynx .raw files (manual and ORIGAMIMS). Furthermore, subsequent visualisation of the precursor and fragment ions in 2D/3D enables a more appropriate interrogation of the relationship between molecular structure and ion dissociation pathways. In addition, by integrating TWIMS calibration into the workflow, the collision cross section data can be directly used to quantify the structural changes occurring during the unfolding and dissociation processes. Moreover, integration of the methodology employed by Allison et al.ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/ncomms9551", "ISBN" : "2041-1723 (Electronic)\r2041-1723 (Linking)", "ISSN" : "2041-1723", "PMID" : "26440106", "abstract" : "The effects of protein-ligand interactions on protein stability are typically monitored by a number of established solution-phase assays. Few translate readily to membrane proteins. We have developed an ion-mobility mass spectrometry approach, which discerns ligand binding to both soluble and membrane proteins directly via both changes in mass and ion mobility, and assesses the effects of these interactions on protein stability through measuring resistance to unfolding. Protein unfolding is induced through collisional activation, which causes changes in protein structure and consequently gas-phase mobility. This enables detailed characterization of the ligand-binding effects on the protein with unprecedented sensitivity. Here we describe the method and software required to extract from ion mobility data the parameters that enable a quantitative analysis of individual binding events. This methodology holds great promise for investigating biologically significant interactions between membrane proteins and both drugs and lipids that are recalcitrant to characterization by other means.", "author" : [ { "dropping-particle" : "", "family" : "Allison", "given" : "Timothy M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Reading", "given" : "Eamonn", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Liko", "given" : "Idlir", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Baldwin", "given" : "Andrew J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Laganowsky", "given" : "Arthur", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "V", "family" : "Robinson", "given" : "Carol", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature communications", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2015" ] ] }, "page" : "8551", "publisher" : "Nature Publishing Group", "title" : "Quantifying the stabilizing effects of protein-ligand interactions in the gas phase.", "type" : "article-journal", "volume" : "6" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "30", "plainTextFormattedCitation" : "30", "previouslyFormattedCitation" : "30" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }30 whereby, protein and protein-ligand stabilities were extracted from gas phase aIM-MS experiments, could provide useful insights which might be utilised to aid conformational sampling techniques. Figure 4. Output from ORIGAMIMS acquisition following ORIGAMIANALYSE mediated aIM-MS of the tetrameric Concanavalin A [4M+20H]20+ ion. The data was acquired using a linear voltage ramp from 4-180 V with a step size of 2 V and 15 s of acquisition per collision voltage (3 SPV and scan time of 5 s) and a total experiment time of 23 minutes. (a) Mass spectrum taken at 4 V showing the presence of only ConA [4M+20H]20+; (b) MS taken from the entire aIM-MS acquisition showing the presence of precursor and fragment ions (primarily monomers); (c) aIM-MS data of all ions and three of the product ions assigned as the [M+10H]10+ (red), [M+11H]11+ (green) and [M+12H]12+ (purple) monomers of ConA overlayed on the aIM-MS heatmap of the [4M+20H]20+ parent ion (blue). The monomeric ions first appear between 58-68 V following the first major unfolding event. Annotations shown in (c) are in the laboratory frame energy (Elab) and corresponding collision voltage units.To demonstrate the aIM-MS overlay method for biomolecules, we show the collisional unfolding of the ConA 20+ tetramer obtained using ORIGAMIMS. Activated IM-MS was performed between 4-180 V with a step size of 2 V and 3 scans per voltage (total of 15 s per collision voltage). Upon disruption of the non-covalent interactions, the first monomeric ions appear. Figure 4a shows the mass spectrum at a collision voltage of 4 V, showing only the precursor ion peak; Figure 4b shows the total mass spectrum obtained for the entire aIM-MS experiment, where the monomeric fragment ions produced by activation are observed in a charge state range of 9+ to 14+ as has been reported previously for non IM tandem MS. Since unfolding and fragmentation occurs in the trap prior to mobility separation, fragment monomeric ions can undergo further unfolding and even MS3. A 2D aIM heatmap for all ions is shown in Figure 4c (top left), whilst an overlay of the 3 dominant monomer product ions along with the tetrameric parent species is shown alongside. The overlay plots are annotated with the collision voltage and corresponding laboratory frame energy (Elab) at which the fragment ion appears. The Elab was calculated using equation 3 where CV is the collision voltage and z is the charge of the ion. Elab=CV×z (3)The ConA parent ion retains a narrow ATD profile and collapses slightly until the collision voltage increases to 58 V (1160 eV, at 20+) where the first unfolding event occurs. As previously reported, collisional unfolding pathways are dependent upon the charge state of the ion;ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/ac101121r", "ISBN" : "0003-2700", "ISSN" : "00032700", "PMID" : "20481443", "abstract" : "Tandem mass spectrometry (MS) of large protein complexes has proven to be capable of assessing the stoichiometry, connectivity, and structural details of multiprotein assemblies. While the utility of tandem MS is without question, a deeper understanding of the mechanism of protein complex dissociation will undoubtedly drive the technology into new areas of enhanced utility and information content. We present here the systematic analysis of the charge state dependent decay of the noncovalently associated complex of human transthyretin, generated by collision-induced dissociation (CID). A crown ether based charge reduction approach was applied to generate intact transthyretin tetramers with charge states ranging from 15+ to 7+. These nine charge states were subsequently analyzed by means of tandem MS and ion mobility spectrometry. Three different charge-dependent mechanistic regimes were identified: (1) common asymmetric dissociation involving ejection of unfolded monomers, (2) expulsion of folded monomers from the intact tetramer, and (3) release of C-terminal peptide fragments from the intact complex. Taken together, the results presented highlight the potential of charge state modulation as a method for directing the course of gas-phase dissociation and unfolding of protein complexes.", "author" : [ { "dropping-particle" : "", "family" : "Pagel", "given" : "Kevin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hyung", "given" : "Suk Joon", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ruotolo", "given" : "Brandon T.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "V.", "family" : "Robinson", "given" : "Carol", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Analytical Chemistry", "id" : "ITEM-1", "issue" : "12", "issued" : { "date-parts" : [ [ "2010" ] ] }, "page" : "5363-5372", "title" : "Alternate dissociation pathways identified in charge-reduced protein complex ions", "type" : "article-journal", "volume" : "82" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "26", "plainTextFormattedCitation" : "26", "previouslyFormattedCitation" : "26" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }26 in the case of the 10+ ion (top right, red), the species appears upon the first unfolding event and the protein retains the ‘original’ unfolded state without further structural rearrangement. The 11+ (bottom left, green) and 12+ (bottom right, purple) ions are also released at the same time, however both species experience further unfolding at 146 V (1600 eV, at 11+) and 120 V (1440 eV, at 12+), respectively. The utilisation of the overlay method and rapid extraction tools can significantly enhance analytical workflows with regard to monitoring relative populations and stabilities of precursor and fragment ions. An immediate candidate for such a protocol is the monitoring of non-covalent binding interactions of cofactors or drug candidates with biomolecules. TWIMS calibrationDetermining collision cross sections from TWIMS measurements requires calibration using appropriate ions with known CCS values;ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/ac1022953", "ISSN" : "1520-6882", "PMID" : "20979392", "abstract" : "Collision cross sections in both helium and nitrogen gases were measured directly using a drift cell with RF ion confinement inserted within a quadrupole/ion mobility/time-of-flight hybrid mass spectrometer (Waters Synapt HDMS, Manchester, U.K.). Collision cross sections for a large set of denatured peptide, denatured protein, native-like protein, and native-like protein complex ions are reported here, forming a database of collision cross sections that spans over 2 orders of magnitude. The average effective density of the native-like ions is 0.6 g cm(-3), which is significantly lower than that for the solvent-excluded regions of proteins and suggests that these ions can retain significant memory of their solution-phase structures rather than collapse to globular structures. Because the measurements are acquired using an instrument that mimics the geometry of the commercial Synapt HDMS instrument, this database enables the determination of highly accurate collision cross sections from traveling-wave ion mobility data through the use of calibration standards with similar masses and mobilities. Errors in traveling-wave collision cross sections determined for native-like protein complexes calibrated using other native-like protein complexes are significantly less than those calibrated using denatured proteins. This database indicates that collision cross sections in both helium and nitrogen gases can be well-correlated for larger biomolecular ions, but non-correlated differences for smaller ions can be more significant. These results enable the generation of more accurate three-dimensional models of protein and other biomolecular complexes using gas-phase structural biology techniques.", "author" : [ { "dropping-particle" : "", "family" : "Bush", "given" : "Matthew F", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hall", "given" : "Zoe", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Giles", "given" : "Kevin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hoyes", "given" : "John", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "V", "family" : "Robinson", "given" : "Carol", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ruotolo", "given" : "Brandon T", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Analytical chemistry", "id" : "ITEM-1", "issue" : "22", "issued" : { "date-parts" : [ [ "2010", "11", "15" ] ] }, "page" : "9557-9565", "title" : "Collision cross sections of proteins and their complexes: a calibration framework and database for gas-phase structural biology.", "type" : "article-journal", "volume" : "82" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "author" : [ { "dropping-particle" : "", "family" : "Bush", "given" : "Matthew F", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "", "id" : "ITEM-2", "issued" : { "date-parts" : [ [ "0" ] ] }, "title" : "Bush CCS Database", "type" : "article-journal" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "36,37", "plainTextFormattedCitation" : "36,37", "previouslyFormattedCitation" : "36,37" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }36,37 the calibration procedure is necessary due to the non-linear relationship between the mobility and drift time of the ions separated by the travelling wave. Since the first release of Waters Synapt instruments, several calibration protocols have been developed, all of which rely upon the use of one or more calibrant ions selected based on the analyte type and molecular weight.ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/nprot.2008.78", "ISSN" : "1750-2799", "PMID" : "18600219", "abstract" : "Here we describe a detailed protocol for both data collection and interpretation with respect to ion mobility-mass spectrometry analysis of large protein assemblies. Ion mobility is a technique that can separate gaseous ions based on their size and shape. Specifically, within this protocol, we cover general approaches to data interpretation, methods of predicting whether specific model structures for a given protein assembly can be separated by ion mobility, and generalized strategies for data normalization and modeling. The protocol also covers basic instrument settings and best practices for both observation and detection of large noncovalent protein complexes by ion mobility-mass spectrometry.", "author" : [ { "dropping-particle" : "", "family" : "Ruotolo", "given" : "Brandon T", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Benesch", "given" : "Justin L P", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sandercock", "given" : "Alan M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hyung", "given" : "Suk-Joon", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "V", "family" : "Robinson", "given" : "Carol", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature protocols", "id" : "ITEM-1", "issue" : "7", "issued" : { "date-parts" : [ [ "2008", "1" ] ] }, "page" : "1139-1152", "title" : "Ion mobility-mass spectrometry analysis of large protein complexes.", "type" : "article-journal", "volume" : "3" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1002/rcm.3737", "ISSN" : "0951-4198", "PMID" : "18816489", "abstract" : "The three-dimensional conformation of a protein is central to its biological function. The characterisation of aspects of three-dimensional protein structure by mass spectrometry is an area of much interest as the gas-phase conformation, in many instances, can be related to that of the solution phase. Travelling wave ion mobility mass spectrometry (TWIMS) was used to investigate the biological significance of gas-phase protein structure. Protein standards were analysed by TWIMS under denaturing and near-physiological solvent conditions and cross-sections estimated for the charge states observed. Estimates of collision cross-sections were obtained with reference to known standards with published cross-sections. Estimated cross-sections were compared with values from published X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy structures. The cross-section measured by ion mobility mass spectrometry varies with charge state, allowing the unfolding transition of proteins in the gas phase to be studied. Cross-sections estimated experimentally for proteins studied, for charge states most indicative of native structure, are in good agreement with measurements calculated from published X-ray and NMR structures. The relative stability of gas-phase structures has been investigated, for the proteins studied, based on their change in cross-section with increase in charge. These results illustrate that the TWIMS approach can provide data on three-dimensional protein structures of biological relevance.", "author" : [ { "dropping-particle" : "", "family" : "Scarff", "given" : "Charlotte a", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Thalassinos", "given" : "Konstantinos", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hilton", "given" : "Gillian R", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Scrivens", "given" : "James H", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Rapid communications in mass spectrometry : RCM", "id" : "ITEM-2", "issue" : "20", "issued" : { "date-parts" : [ [ "2008", "10" ] ] }, "page" : "3297-304", "title" : "Travelling wave ion mobility mass spectrometry studies of protein structure: biological significance and comparison with X-ray crystallography and nuclear magnetic resonance spectroscopy measurements.", "type" : "article-journal", "volume" : "22" }, "uris" : [ "" ] }, { "id" : "ITEM-3", "itemData" : { "DOI" : "10.1255/ejms.947", "ISSN" : "1469-0667", "PMID" : "19423898", "abstract" : "Detailed knowledge of the tertiary and quaternary structure of proteins and protein complexes is of immense importance in understanding their functionality. Similarly, variations in the conformational states of proteins form the underlying mechanisms behind many biomolecular processes, numerous of which are disease-related. Thus, the availability of reliable and accurate biophysical techniques that can provide detailed information concerning these issues is of paramount importance. Ion mobility spectrometry (IMS) coupled to mass spectrometry (MS) offers a unique opportunity to separate multi-component biomolecular entities and to measure the molecular mass and collision cross-section of individual components in a single, rapid (</= 2 min) experiment, providing 3D- architectural information directly. Here we report a method of calibrating a commercially available electrospray ionisation (ESI)-travelling wave ion mobility spectrometry (TWIMS)-mass spectrometer using known cross-sectional areas determined for a range of biomolecules by conventional IMS-MS. Using this method of calibration, we have analysed a range of proteins of differing mass and 3D architecture in their native conformations by ESI-TWIMS-MS and found that the cross-sectional areas measured in this way compare extremely favourably with cross-sectional areas calculated using an in-house computing method based on Protein Data Bank NMR-derived co-ordinates. This not only provides a high degree of confidence in the calibration method, but also suggests that the gas phase ESI- TWIMS-MS measurements relate well to solution-based measurements derived from other biophysical techniques. In order to determine which instrumental parameters affect the ESI-TWIMS-MS cross-sectional area calibration, a systematic study of the parameters used to optimise TWIMS drift time separations has been carried out, observing the effect each parameter has on drift times and IMS resolution. Finally, the ESI-TWIMS-MS cross-sectional area calibration has been applied to the analysis of the amyloidogenic protein beta(2)-microglobulin and measurements for three co-populated conformational families, present under denaturing conditions, have been made: the folded, partially unfolded and unfolded states.", "author" : [ { "dropping-particle" : "", "family" : "Smith", "given" : "David P", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Knapman", "given" : "Tom W", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Campuzano", "given" : "Iain", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Malham", "given" : "Richard W", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Berryman", "given" : "Joshua T", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Radford", "given" : "Sheen E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ashcroft", "given" : "Alison E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "European journal of mass spectrometry (Chichester, England)", "id" : "ITEM-3", "issue" : "2", "issued" : { "date-parts" : [ [ "2009", "1" ] ] }, "page" : "113-130", "title" : "Deciphering drift time measurements from travelling wave ion mobility spectrometry-mass spectrometry studies.", "type" : "article-journal", "volume" : "15" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "19,38,39", "plainTextFormattedCitation" : "19,38,39", "previouslyFormattedCitation" : "19,38,39" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }19,38,39 As with many analytical measurements, their accuracy is determined by the selection of appropriate calibrants and careful measurements. Consequently, the experimental conditions (particularly regarding IM and ToF parameters) for both the unknown and calibrant ions must be kept constant. Upon loading of the calibration files and selecting which calibrants were used, ORIGAMIANALYSE loads the mass spectrum, extracts drift times of each charge state and annotates each ion with CCS values from the supplied formatted CCS database. In cases where calibrant species have a single well-defined conformation, a single Gaussian peak is fitted to the ATD to retrieve the centroid drift time to be used in the CCS calibration; in more complicated cases, where the ion adopts multiple conformations, ORIGAMIANALYSE fits multiple Gaussian peaks to the ATD and lets the user select the most appropriate peak to build the calibration curve. Each calibration curve can be generated by using single or multiple files, and can be modified at any point by selecting or deselecting species. The user can also provide calibration parameters from elsewhere (i.e. from manually calibrated datasets). The calibration curves obtained in this way can be applied to a single extracted ion, list of ions or saved to MassLynx files and exported in a .csv format. The CCS calibration workflow is shown in Figure S6. Interactive datasetsVisualisation of scientific research is typically accomplished using static images embedded in documents (i.e. PDF) or on websites. While a ‘picture is worth a thousand words’ is a well-known expression, the rigid nature of published figures can hamper a clear understanding of scientific research and often is not enough to fully convey the story. With this in mind, ORIGAMIANALYSE has built-in functionality to export any of the figures (1D, 2D and 3D) in an interactive format (with active controls, i.e. zoom, rotation, selection, annotation and more) that can be embedded on websites or shared as individual documents (i.e. as supplementary information in scientific publication). In order to export normally static-images in an interactive format, the plots are made using the Bokeh Python package, which saves data in a JSON (JavaScript) format, embedded into a .html file. Each individual figure can be appropriately annotated (i.e. title, figure caption, experimental conditions) in a HTML-rich format. The type of interactivity associated with each figure can be selected by the user, as well as the type of built-in annotations. The output format takes advantage of the modern internet browsers, hence can be viewed in any internet browser and is therefore device independent. The availability of interactive scientific figures is to increase the transparency, aid understanding of complex datasets and give the reader (and the user) an easily accessible way to visualise, store and share, in this case, aIM-MS datasets. An example of such a file is shown in Supplementary File 2.ConclusionsORIGAMI provides an efficient means of data acquisition and a more in-depth analysis of TWIMS acquired aIM datasets. The automation of the collision voltage ramp can be utilised to employ aIM in high-throughput environments, effectively reducing acquisition, data handling and analysis timescales. While we present data acquired with ORIGAMIMS and then analysed by ORIGAMIANALYSE, the analysis module can be used as a standalone program on manually acquired datasets but also on non-activated IM-MS MassLynx files. We have shown applications most relevant to the field of structural biology, however the core functionality is not limited to large biomolecules; application for small molecule analysis where aIM-MS can be insightful is another area where this enhanced workflow may be useful.ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/acs.analchem.6b04998", "ISSN" : "0003-2700", "author" : [ { "dropping-particle" : "", "family" : "Gray", "given" : "Christopher John", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Schindler", "given" : "Baptiste", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Migas", "given" : "Lukasz G.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Picmanova", "given" : "Martina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Allouche", "given" : "Abdul-Rahman", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Green", "given" : "Anthony P.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mandal", "given" : "Santanu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Motawia", "given" : "Mohammed Saddik", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "S\u00e1nchez-P\u00e9rez", "given" : "Raquel", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bjarnholt", "given" : "Nanna", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "M\u00f8ller", "given" : "Birger Lindberg", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rijs", "given" : "Anouk M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Barran", "given" : "Perdita E.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Compagnon", "given" : "Isabelle", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Eyers", "given" : "Claire E.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Flitsch", "given" : "Sabine L.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Analytical Chemistry", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2017" ] ] }, "page" : "4540-4549", "title" : "Bottom-up elucidation of glycosidic bond stereochemistry", "type" : "article-journal", "volume" : "89" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "40", "plainTextFormattedCitation" : "40", "previouslyFormattedCitation" : "41" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }40 Since the software is open-source, written in Python and utilises pre-existing Driftscope libraries, ORIGAMI is extensible and we envisage additional functionality can be readily added to both ORIGAMIMS and ORIGAMIANALYSE. AcknowledgementsL.G.M. would like to thank Emmy Hoyes for introduction to the wonderful world of WREnS scripting and Keith Richardson for initial help in the analysis of MassLynx .raw files (Waters Corp., Manchester). We gratefully acknowledge BBSRC and MRC for support of this work in studentships to L.G.M. and A.F. respectively. This work was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) by grants, BB/L015048/1 and BB/M017702/1. We also thank the British Mass Spectrometry Society (BMSS) and University of Manchester for continued support of our research. Author contributionsL.G.M. and B.B. designed ORIGAMIMS whilst L.G.M. wrote both software components. L.G.M. and A.F. carried out the mass spectrometry experiments. L.G.M. analysed the data and wrote the first draft of the manuscript. All authors contributed towards final editing of the manuscript under the supervision of P.E.B and with additional well-meaning kind advice from reviewers.ReferencesADDIN Mendeley Bibliography CSL_BIBLIOGRAPHY 1.Lapthorn, C. et al. How useful is molecular modelling in combination with ion mobility mass spectrometry for ‘small molecule’ ion mobility collision cross-sections? Analyst 140, 6814–6823 (2015).2.Warnke, S. et al. Protomers of benzocaine: Solvent and permittivity dependence. J. Am. Chem. Soc. 137, 4236–4242 (2015).3.Voronina, L. et al. Conformations of prolyl-peptide bonds in the bradykinin 1-5 fragment in solution and in the gas phase. 138, 9224–9233 (2016).4.Beveridge, R. et al. Mass spectrometry locates local and allosteric conformational changes that occur on cofactor binding. Nat. Commun. 7, 12163 (2016).5.Liu, Y., Cong, X., Liu, W. & Laganowsky, A. Characterization of Membrane Protein – Lipid Interactions by Mass Spectrometry Ion Mobility Mass Spectrometry. 579–586 (2017). doi:10.1007/s13361-016-1555-16.Clemmer, D. E., Hudgins, R. R. & Jamold, M. F. Naked Protein Conformations: Cytochrome. 10141–10142 (1995).7.Quintyn, R. S., Zhou, M., Yan, J. & Wysocki, V. H. Surface-Induced Dissociation Mass Spectra as a Tool for Distinguishing Different Structural Forms of Gas-Phase Multimeric Protein Complexes. Anal. Chem. acs.analchem.5b03441 (2015). doi:10.1021/acs.analchem.5b034418.Pacholarz, K. J. et al. Hybrid Mass Spectrometry Approaches to Determine How L-Histidine Feedback Regulates the Enzyzme MtATP-Phosphoribosyltransferase. Structure 25, 1–9 (2017).9.Rabuck, J. N. et al. Activation state-selective kinase inhibitor assay based on ion mobility-mass spectrometry. Anal. Chem. 85, 6995–7002 (2013).10.Harvey, S. R. et al. Small-molecule inhibition of c-MYC:MAX leucine zipper formation is revealed by ion mobility mass spectrometry. J. 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Ion Mobility-Mass Spectrometry and Collision Induced Unfolding Rapidly Detect Subtle Differences in Antibody Glycoforms Therapeutic Antibodies?: Impact and Opportunity. (2016).16.Mason, E. A. & McDaniel, E. W. Transport properties of ions in gases. (Wiley, New York, 1988).17.Giles, K. et al. Applications of a travelling wave-based radio-frequency-only stacked ring ion guide. Rapid Commun. Mass Spectrom. 18, 2401–2414 (2004).18.Giles, K., Williams, J. P. & Campuzano, I. Enhancements in travelling wave ion mobility resolution. Rapid Commun. Mass Spectrom. 25, 1559–1566 (2011).19.Ruotolo, B. T., Benesch, J. L. P., Sandercock, A. M., Hyung, S.-J. & Robinson, C. V. Ion mobility-mass spectrometry analysis of large protein complexes. Nat. Protoc. 3, 1139–1152 (2008).20.Niu, S. & Ruotolo, B. T. Collisional unfolding of multiprotein complexes reveals cooperative stabilization upon ligand binding. Protein Sci. 24, 1272–1281 (2015).21.Eschweiler, J. D., Martini, R. M. & Ruotolo, B. T. Chemical Probes and Engineered Constructs Reveal a Detailed Unfolding Mechanism for a Solvent-Free Multidomain Protein. J. Am. Chem. Soc. jacs.6b11678 (2016). doi:10.1021/jacs.6b1167822.Shelimov, K. B., Clemmer, D. E., Hudgins, R. R. & Jarrold, M. F. Protein Structure in Vacuo?: Gas-Phase Conformations of BPTI and Cytochrome c. J. Am. Chem. Soc. 7863, 2240–2248 (1997).23.Clemmer, D. E. & Jarrold, M. F. Ion Mobility Measurements and their Applications to Clusters and Biomolecules. J. Mass Spectrom. 32, 577–592 (1997).24.Helden, G. Von, Gotts, N. & Bowers, M. T. Experimental evidence for the formation of fullerenes by collisional heating of carbon rings in the gas phase. Nature 363, 60–63 (1993).25.Zhong, Y., Han, L. & Ruotolo, B. T. Collisional and Coulombic Unfolding of Gas-Phase Proteins: High Correlation to Their Domain Structures in Solution. Angew. Chem. Int. Ed. Engl. 53, 1–5 (2014).26.Pagel, K., Hyung, S. J., Ruotolo, B. T. & Robinson, C. V. 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