Perspectives of peptide chemistry and combinatorial ...



Perspectives of peptide chemistry and combinatorial chemistry for proteomics

Ferenc Hudecz

Research Group of Peptide Chemistry at Eötvös L. University,

Hungarian Academy of Sciences,

1518 Budapest 112, P.O.B. 32, Hungary

MUCIN-2 EPITOPE PEPTIDES: IDENTIFICATION BY COMBINATORIAL CHEMISTRY AND MASS SPECTROMETRY

At the beginning of the 20th century Emil Fischer published the preapartion of glycylglycine by partial hydrolysis of diketopiperazine in 1901. The modern peptide chemistry began in 1932 by the introduction of benzyloxycarbonyl protecting group by Bergmann and Zervas. In 1963 Merrifield announced the first the solid phase peptide synthesis of a tetrapeptide. The invention of combinatorial peptide synthesis by Furka in 1988 led to the discovery of new together with the development of parallel and multiple synthesis strategies led to a revolutionary progress in the field of peptide science. Chemistries for synthesis, techniques for purification (e.g. HPLC, electrophoresis) and sophisticated methods for elucidation of primary and 3D structure (e.g. mass spectrometry, NMR) now are available for addressing problems raised by proteome research. Identification of proteins with unknown or partially known function by proteomics requires not only data on the structure of the intact molecule, but also localization of domains, segments responsible for the binding or biological activity. For example by the design of appropriate combinatorial mixtures or overlapping set of peptides relevant segments can be determined. Peptide chemistry will not only contribute to the understanding the machinery of cell in health and disease, but also could serve as a tool in drug-development on validated target proteins or in even targeting drugs into defined population of cells. This lecture will outline some results obtained in the Budapest laboratory in the field of identification of epitopes of mucin glycoproteins, development and application of synthetic antigens in diagnostics (e.g. rheumatoid arthritis, Herpes simplex virus infection) and vaccine design (e.g. Alzheimer disease, M. tuberculosis) and of targeting of tumour cells by peptide carriers.

New Microfluidic-Mass Spectrometry Technologies for High Performance Proteomics

First progress report (10/2001 – 11/2002)

University of Konstanz (M. Przybylski), Curie-Institute Paris (J.L. Viovy), University of Verona (P.G. Righetti), Ecole Polytechnique Federale Lausanne (H. Girault), Diagnoswiss SA (J. Rossier), Ademtech SA (B. Plichon)

ABSTRACT

The main scientific objective of the project is to promote and facilitate proteomics studies by developing new technologies for the analysis of proteins in complex mixtures, that are complementary to conventional 2-dimensional gel electrophoresis. To reach this goal, the design, analytical development and industrial fabrication of a complete platform is pursued comprising a new generation of automated micro- and nano-scale proteomics systems for isolation, purification and structural characterisation of proteins from complex biological mixtures. Key experimental approaches and methods are (i) the development of new pre-fractionation microelectrolysers in miniaturised format to fit the requirements of microfluidic systems; (ii) synthesis of new wall-coated polymers for use in fused silica capillaries, and of thermosensitive matrices suitable for proteomics microchips; (iii) design and fabrication of polymeric microchips with performance characteristics, suitable for general microfluidic applications to high performance proteomics; and (iv) instrumental development and adaptation of high resolution, nanoelectrospray mass spectrometry (MS), particularly Fourier transform ion cyclotron resonance (FTICR-MS) is pursued, suitable for use as a high performance chip-MS platform, and for proteomics applications with high resolution and high selectivity. The major results of the first period of the project have been the development of a new miniaturised multi-compartment electrolyser for protein pre-fractionation, and new wall-coated polymers for high performance separation in fused silica capillaries; the successful development and semi-industrial production of plasma etched polymer microchips suitable for nanospray-MS. A high performance yet robust thin-chip electrospray-MS system has been developed and has been fully compatible with high resolution-FTICR mass spectrometry. Major advantages of this chip system such as high sensitivity, long spraying times and low background contamination have been demonstrated in initial proteomics applications.

Disposable Nanospray Tips for Electrospray Ionisation

Mass Spectrometry

J. S. Rossier(1), P. Michel(1), N. Lion(2), V. Gobry(2), H.H. Girault(2) and F. Reymond(1)

(1) DiagnoSwiss SA, Rte de l’Ile-au-Bois 2, CH-1870 Monthey (Switzerland)

(2) Laboratoire d’Electrochimie Physique et Analytique, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne (Switzerland)

Electrospray ionisation (ESI) has become over the last 10 years a major tool for the quantification of pharmaceutical compounds in biological fluids and for peptide and protein characterization [1-2]. In proteomic applications, very small amounts of proteins and peptides are analysed either using liquid chromatography at nanoliter flow rates or by infusion with nanoelectrospray of 1D- or 2D-gel spots after digestion with trypsin. However, nanoelectrospray is currently not automated and samples cannot be analysed in an unattended mode, which affects dramatically sample throughput. In addition, standard electrospray needles require long washing steps to prevent sample cross-contamination. Disposable miniaturized electrospray sources are thus very attractive to overcome these limitations.

In this communication, we will indeed present plasma etched polyimide nanospray chips with integrated electrodes [3] that have been designed as thin film nanoelectrospray tips for protein analysis by electrospray ionisation mass spectrometry (ESI-MS) in an infusion mode [4-5]. As will be shown here, characterization of these mass produced nanospray chips demonstrates good spray stability, outstanding on/off spray switching, good sensitivity as well as easy automation abilities.

Work has also been achieved in order to integrate new functions in these nanospray chips. In particular, antibodies have been immobilized on the micro-channel walls in order to specifically capture a protein of interest before electrospray sampling and MS determination. Similarly, a PVDF membrane has been introduced at the inlet port of the micro-channel so as to desalt the sample solution prior to injection into the MS (results not shown here, but presented in ref. [6]). On the other hand, preliminary characterisation of MS sampling of aqueous solutions using a sheath liquid has been performed using a duo-channel microchips that enables mixing of the sample and sheath liquid solutions directly in the Taylor cone.

The versatility of our nanospray chips, their automation and the integration of supplementary functions like desalting or affinity capture render them specially well-suited for the accurate analysis proteins in high-throughput and without cross-contamination, which a key aspects in proteomics applications.

References

1) G. Hopfgartner, C. Husser, M. Zell, Ther. Drug Monit. 2002, 24, 134-143; 2) M. Mann, R.C. Hendrickson, Annu. Rev. Biochem. 2001, 70, 437-373; 3) J. S. Rossier, F. Reymond, P.E. Michel, Electrophoresis 2002, 23, 858-867; 4) V. Gobry, J. van Oostrum, M. Martinelli, T. Rohner, F. Reymond, J.S. Rossier, H.H. Girault, Proteomics 2002, 2, 405-412; 5) J.S. Rossier, N. Youhnovski, N. Lion, E. Damoc, F. Reymond, H.H. Girault, M. Przybylski, Angw. Chemie, 2003, 115, 55-60; 6) N. Lion, V. Gobry, H. Jensen, J.S. Rossier, H.H. Girault, Electrophoresis, 2002, 23, 3583-3588.

Design and Fabrication of Plasma Etched Polymer Micro-chips for High-performance Protein Analysis

P. Michel, P. Morier, F. Reymond and J.S. Rossier

DiagnoSwiss SA, Rte de l’Ile-au-Bois 2, CH-1870 Monthey (Switzerland)

Miniaturization of analytical systems has played an important role in the development of fast analytical tools using very small amounts of sample. Intensive efforts have thus been placed in the design of miniaturised complete laboratory systems mainly devoted to capillary electrophoresis and electrochromatography [1-2]. If most systems were first produced in glass substrates, polymer micro-chips have gained increasing interest due to the simplified manufacturing procedures compared to glass (notably for the sealing of the micro-structures) which also enables mass production of low cost disposable devices in a large variety of materials [3].

In this domain, DiagnoSwiss’ micro-chips have the particularity to be thin polymer foils that comprise micro-channels with integrated gold micro-electrodes and that are sealed by lamination of a second polymer layer [4-5]. One key feature of the industrial production process is the direct introduction of the micro-electrodes during the fabrication, thereby avoiding cumbersome and expensive post-processing and offering a novel type of electrochemical biosensors.

In this work, we present the plasma etching process used to produce our micro-systems, as well as the development steps achieved so far to manufacture disposable tips for electrospray ionisation mass spectrometry (ESI-MS). We will also show here how the chips are designed and what are the geometrical characteristics of the micro-channels, the electrodes and the spray tips. Special attention is also given here to the development of duo-channel chips that are mainly devoted to the mixing of a sample solution with a sheath liquid used to generate the electrospray.

The main difficulty in this special fabrication process yet remains the precise cutting of the micro-channel extremity to produce the electrospray tip, which has to be very precise, reproducible and compatible with the sealing process used. Ways to overcome this issue will be investigated in the next development phase, so as to obtain a complete production line of nanospray chips that will meet all the requirements for stable spray and controlled flow rate.

The development of these industrial ESI chips showed that the geometrical parameters of the micro-channels and integrated electrodes are controlled efficiently and that plasma etching allows to create versatile three-dimensional micro-systems in thin polyimide foils that already possess all the features required for fast and parallel analysis in a contamination-free environment., notably for high performance proteomics [6].

References

1) J.P. Kutter, Trends Anal. Chem., 2000, 19, 352-363; 2) G.J.M. Bruin, Electrophoresis, 2000, 21, 3931; 3) S.A. Soper, S.M. Ford, S.L.M.R. Qi, K. Kelly, M.C. Murphy, Anal. Chem., 2000, 72, 642A; 4) J. S. Rossier, F. Reymond, P.E. Michel, Electrophoresis 2002, 23, 858; 5) V. Gobry, J. van Oostrum, M. Martinelli, T. Rohner, F. Reymond, J.S. Rossier, H.H. Girault, Proteomics 2002, 2, 405; 6) J.S. Rossier, N. Youhnovski, N. Lion, E. Damoc, F. Reymond, H.H. Girault, M. Przybylski, Angw. Chemie, 2003, 115, 55.

Pre-fractionation of proteins via multicompartment

electrolyzers and fraction analysis via polymer microfluidic chips coupled to mass spectrometers

Annalisa Castagna1, Pier Giorgio Righetti1, Niels Lion2, Hubert H. Girault2,

Joël S. Rossier3, Frédéric Reymond3

1University of Verona, Department of Agricultural and Industrial Biotechnologies,

37134 Verona, Italy

2Ecole Polytechnique Fédérale de Lausanne, Laboratoire d’Electrochime physique et Analytique, 1015 Lausanne, Switzerland

3Diagnoswiss S.A., Rte de l’Ile-au-Bois 2, 1870 Monthey, Switzerland

Pre-fractionation is a must in proteome analysis, due to the extreme complexity of an entire cell protein lysate [1]. One method recently proposed consists in adopting multicompartment electrolyzers (MCE), endowed with isoelectric membranes of well-defined pI values, able to collect in a given chamber a certain protein populations having pIs encompassing the pI values of said membranes [2, 3]. MCEs can provide narrow pI fractions, down to only 0.5 pH units, fully compatible with any focusing technique, since such fractions are desalted by the focusing process occurring in the MCE instrument. In the recent years, new polymer microfluidic chips have been developed, in which capillary electrophoretic separations of protein mixtures (of reduced complexity) as well as ELISA-type immunological assays can be conducted in short analysis times [4, 5]. In turn, such chips can be coupled to a mass spectrometer (MS), for identification of the eluted protein fractions [4, 6]. We have thus explored the compatibility of narrow pI cuts, as obtained with the MCE instrumentation, with subsequent affinity separation in chips or characterization in disposable nanospray chips/MS. Narrow-pI fractions of standard protein solutions, obtained both in the acidic and in basic pH intervals, have been obtained via MCE fractionation and subjected to further analysis in the microchip/MS systems. Attempts to analyze complex protein mixtures using the above methodology will also be presented here and evaluated.

[1] Righetti, P.G., Stoyanov, A., Zhukov, M.: The Proteome revisited: Theory and Practice of the Relevant Electrophoretic Steps. Elsevier, Amsterdam, 2001, 400 pp.

[2] Herbert, B., Righetti, P.G.: A turning point in proteome analysis: sample pre-fractionation via multicompartment electrolyzers with isoelectric membranes. Electrophoresis 21 (2000) 3639-3648.

[3] Righetti, P.G., Castagna, A., Herbert, B.: Prefractionation techniques in proteome analysis. Anal. Chem. 73 (2001) 320A-326A.

[4] Rossier, J.S., Reymond, F., Michel, P.E.: Polymer microfluidic chips for electrochemical and biochemical analysis. Electrophoresis 23 (2002) 858-867.

[5] Rossier, J.S., Girault, H.H.: Enzyme Linked Immunosorbent Assay on a Microchip with Electrochemical Detection, Lab Chip 1 (2001) 153-157.

[6] Gobry, V., Van Oostrum, J., Martinelli, M., Rohner, Reymond, F., Rossier J.S., Girault, H.H.: Microfabricated Polymer Injector for Direct Mass Spectrometry Coupling, Proteomics, 2002, 2, 405-412.

Approaches and applications of Fourier transform-ICR mass spectrometry to proteome analysis with high resolution and high selectivity

Michael Przybylski

Department of Chemistry, Laboratory of Analytical Chemistry,

University of Konstanz, D-78457 Konstanz, Germany

The development of efficient "soft ionisation" methods in the last years has provided the basis for the molecular characterisation of biopolymers by mass spectrometry. In contrast to previous limitations in the molecular weight range amenable, electrospray ionisation (ESI-MS) and matrix assisted laser desorption ionisation (MALDI-MS) have provided access to biopolymers > 100 kDa. The recent development of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry enabled a breakthrough for the high resolution mass spectrometric structure analysis of biopolymers using both ESI and MALDI ionisation [1]. Present studies in our laboratory in bioanalytical applications of FT-ICR mass spectrometry [2] focus on (i) the analysis of complex peptide mixtures in proteome studies and in combinatorial mixtures; and (ii) the identification of antigenic determinant structures of mono- and polyclonal antibodies using the mass spectrometric epitope mapping method developed in our laboratory [3,4]. In combination with 2D gel electrophoresis the high (sub-ppm) mass determination accuracy and isotopic fine structure by FT-ICR-MS provide particular advantages for the identification of proteins with medium and low abundance; for such applications microchip-ESI-approaches have been recently adapted to FT-ICR-MS.

Major goals of new application areas of FT-ICR-MS are (i) the structure analysis of cell surface proteins and their interaction molecules; (ii) epitope elucidation of antigen target proteins in autoimmune diseases; and (iii) high resolution sequence determinations and characterisation of structure modeifications of target proteins in proteome analysis. These applications are integrated in several research projects and interdisciplinary collaborations at the Departments of Chemistry and Biology of the University of Konstanz (i.a., DFG-project "high resolution biopolymer structure analysis"; DFG-priority programme "Cellular mechanisms of Alzheimers`disease"; EU-project "New microfluidic-mass spectrometry technologies for high performance proteomics"; "Competence centre for proteome analysis" at the University of Konstanz), and the initiation of a European Research Training Centre for High Performance Mass Spectrometry. Recent applications of FT-ICR-MS to neuro-proteomics, interactions of target proteins for Alzheimer`s disease (amyloid precursor proteins & presenilins), and analysis of target antigens for auto-immune diseases will be discussed. In these studies a new approach ("affinity proteomics") has been developed with which FT-ICR-MS is also providing unprecedented identification selectivity for proteins from complex biological mixtures. [5]

[1] A.G. Marshall (1998) Mass Spectrom. Rev. 17, 1.

[2] M. Przybylski et al. (1998) in "New Methods for the Study of Biomolecular Complexes", (W. Ens, ed.), Kluwer Acad. Publ., Amsterdam, 17-43.

[3] M. Przybylski (1995), Adv. Mass Spectrom. 13, 257-283.

[4] M. Macht, W. Fiedler, K. Kürzinger, M. Przybylski (1996) Biochemistry 35, 15633-15638.

[5] M. Kohlmann, M. Macht, S. Deininger, A. Marquardt, M. Przybylski (2001) Proteomics, in press.

[6] M. Przybylski, M.O. Glocker (1996) Angew. Chem. Int. Ed. Engl.35, 806-826.

[7] S. Buehler, J. Michels, S. Wendt, A. Rueck, D. Brdiczka, W. Welte, M. Przybylski (1998) PROTEINS 2, 63-73.

[8] T.A. Fligge, C. Reinhardt, C. Harter, T. Wieland, M. Przybylski (2000) Biochemistry 39, 8491-8496.

[9] S.H. Bauer, X. Zhang, W.V. Dongen, M. Claeys, M. Przybylski (1999) Anal. Biochem. 274, 69-80.

New self-organising matrix materials for

high performance proteomics

Jean Louis Viovy

Laboratoire de Physico-Chimie, Institut Curie, France

The talk will report the main advancements on the project performed in Paris, in the first 18 Months of the project. The general aim of the Paris team is to develop new technological bricks for protein and peptides separation, to be integrated into an on-line microchannel separator, usable as a front end injector for MS.

The first line of research concern the development of new strategies and matrices for the separation of peptides in microchannels. A fundamental study of the factors affecting size fractionation was performed, and new molecular architectures based on the results are being prepared. Also, a strategic effort for direct on-chip fluorescent detection of proteins and peptides was launched, in collaboration with the Pharmaceutical Faculty in Chatenay-Malabry, and encouraging first results are coming out.

The second line is the development of an on-chip microreactor for tryptic digest, to be hyphenated to ESI (collaboration with Lausanne’s group) and FTICR-MS (collaboration with Konstanz) This reactor is based on an original strategy using reversibly self-organizing magnetic beads. Numerous bead fabrication and functionalization strategies were developed, in collaboration with Ademtech and University of Pardubice (see posters by Bilkova et al.) and validated. On the instrumental side, a prototype of microchip magnetic based microreactor was constructed. The beads were tested both in bulk and in the microreactor, showing much faster digestion in the microreactor. Reasons for this improvement will be discussed.

The third line uses the same magnetic beads immobilization strategies, for prion proteomics, in collaboration with Prof. Righetti’s group in Verona.

The perspectives of the project will be discussed, in particular:

-Further improvements of magnetic beads, in collaboration with Ademtech and U. Pardubice

-Use of the new functionalized beads for epitope mapping, in collaboration with Profs Przybylski and Hudecz groups

-Improvement of prion immobilization strategies (reduction of non-specific capture), in collaboration with Pardubice and Verona

-Hyphenation of tryptic digest with ESI (coll. with Lausanne and Brno) and FT-ICR-MS (coll. Konstanz)

-further progress on peptide separation, and integration of separation and tryptic digest on a single chip, towards a µTAS.

Design and development of microfluidic chip systems

for high performance proteomics

Hubert H. Girault

Laboratoire d’Electrochimie Physique et Analytique,

Ecole Polytechnique Fédérale de Lausanne

CH1015 Lausanne, Suisse

Hubert.girault@epfl.ch

We present herein the ongoing developments about the coupling of microfluidic systems with electrospray ionization mass spectrometers. There are mainly two research axis: first, based on the electrochemical behavior of the polymer nanospray, an electrochemical tagging of cysteine residues was developed [1]. Hydroquinone is added to the peptide or protein mixture and then converted into benzoquinone on the nanospray electrode. The benzoquinone thus reacts specifically with free cysteine residues to form an adduct that can be further reduced. The native species and adducts give rise to two distributions in the mass spectrum, which allows the counting of free cysteines. Besides the characterization of substituted hydroquinones as other possible tags and detailed investigation of the effect of design and chemical parameters on the tagging efficiency [2, 3], the possibility to apply this on-line tagging approach in peptide mass fingerprinting experiments is presented.

The second axis is the integration of sample preparation functionalities into MS microchips: a polyvinylidene fluoride membrane is integrated at the inlet of the microchannel, which allows the retention of hydrophobic species such as peptides and proteins. Unretained compounds such as salts and co-solvents can then be washed out, and peptides and proteins directly eluted by the spraying solution [4]. Two typical experiments are presented: first, the alkylation of cysteine residues in (-lactoglobulin A could be performed in aqueous conditions, therefore on-chip desalted and analyzed by mass spectrometry [5]. This kind of application paves the way for on-chip protein structural studies. The second typical experiment is the analysis of the tryptic digest of microtubule-associated protein Tau on the FT-ICR-MS.

EN.REFLIST

High-Performance Proteomics Using a Thin-Chip

Micro-spray System and Fourier transform-ICR

Mass Spectrometry

R. Weber1, E. Damoc1, J.S. Becker1, N. Youhnovski1, N. Lion2, J. Rossier3,

F. Reymond3, H. Girault2, and M. Przybylski1

1 Analytical Chemistry, Department of Chemistry, University of Konstanz,

78467 Konstanz, Germany

2 Laboratoire d’Electrochimie, EPFL-DC, 1015 Lausanne, Switzerland

3 DiagnoSwiss SA, Rte de l’Ile au bois 2, c/o Cimo S.A., 1870 Monthey, Switzerland

High resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) in combination with efficient “soft” ionisation methods (MALDI, ESI) has proved to be a powerful tool in proteome analysis. For high-throughput analysis (e.g. screening of combinatorial libraries and complex protein mixtures from natural sources), automated pre-fractionation and sample handling is desired.

The microchip system we present here is a further step in this direction. It has already been shown that using this chip micro-spray system in combination with FT-ICR MS we were able to detect 18 phosphoylation sites in Tau protein, one of the key proteins in Alzheimer’s Disease (AD) [1].

Now, we present the integration of a desalting device in the microchip [2]. This has shown to be a very useful tool, as high salt concentration have a negative influence on spray stability and the quality of the obtained mass spectra. The digestion of Tau protein was carried out in solution using Promega trypsin. Subsequently, this NH4HCO3-containing solution was directly applied on the chip, passing a PVDF membrane for desalting. By varying the distance between chip and capillary entrance of the mass spectrometer and the capillary exit voltage between 30 – 70 V, we obtained excellent spectra. It was possible to base-line separate two peaks differing in their masses by 0.02 Da.

In a further example, we show that additional to this high resolution the microchip system also provides high sensitivity and high accuracy. Therefore, we analysed a solution with a mixture of standard peptides (c = 2 pmol/µl). In addition to a high resolution (m/Δm = 55000), a high accuracy of mass determination (Δm = 1.2 ppm) was obtained.

Hence, the aims of future projects are the integration of further micro-analytical systems in the chip, e.g. a capillary electrophoresis (CE) unit, and the immobilisation of proteases such as trypsin on magnetic beads. Those projects are carried out together with our co-operation partners DiagnoSwiss (Lausanne), Ademtech (Paris), and Z. Bilkova (University of Pardubice, Czech Republic), respectively.

[1] J. Rossier, N. Youhnovski, J.S. Becker, N. Lion, E. Damoc, F. Reymond, H.H. Girault, M. Przybylski, Thin-chip micro-spray system for high performance Fourier-transform ion cyclotron resonance mass spectrometry of biopolymers, Angew. Chem. Int. Ed. 2003, 42, 53 – 58

[2] J. Rossier, F. Reymond, P.E. Michel, Polymer micro-fluidic chips for electrochemical and biochemical analysis, Electrophoresis 2002, 23, 858 – 867

High-performance affinity proteomics using

chip-microspray systems: analytical development

and applications to vaccine-epitope elucidation of neurodegenerative target proteins

E. Damoc1, R. Cecal1, X. Tian1, A. Marquardt1, M. Manea1, R.Stefanescu1, G. Mezo2,

A. Castagna3, Z. Bilkova4, F. Reymond5, J. Rossier5, J.L. Viovy4, F. Hudecz2,

P.G. Righetti3 and M. Przybylski1

1University of Konstanz, Department of Analytical Chemistry, 78457 Konstanz, Germany

2Research Group of Peptide Chemistry, Hungarian Academy of Sciences,

Eötvös L. University, H-1518, Budapest, Hungary

3University of Verona, Department of Agricultural and Industrial Biotechnologies,

Verona, Italy

4Laboratoire de Physico-Chimie, Institut Curie, Paris, France

5DiagnoSwiss SA, Monthey, Switzerland

The methodology for identification of specific affinity-bound proteins employed in the present study is based on the identification of epitope sequences due to the observation that an antibody will protect the binding site(s) of a bound peptide or protein antigen from proteolytic cleavage. This characteristic feature of antigen-antibody complexes has been employed for the development of a general mass spectrometric approach for the identification of protein epitopes, as initially shown by combining limited proteolytic cleavage of intact immune complexes (epitope excision) with mass spectrometric peptide mapping [1]. Whereas initial applications of mass spectrometric epitope mapping have been performed on small sequence epitopes such as from pure polypeptides, several recent studies have shown that large, native proteins including conformational epitopes can also be successfully investigated. Several bioanalytical applications have ascertained the mass spectrometric epitope mapping / epitope excision approach as a powerful tool.

In the present study, epitope identification of antibodies against the heart muscle protein Troponin T (TnT) has been employed in an "affinity proteomics" approach for direct protein identification from complex biological material [2]. Other examples are the identification of a specific epitope from the Alzheimer's amyloid precursor protein, and the elucidation of an amyloid plaque-specific epitope which directly provides a lead structure for vaccine development against Alzheimer's disease.

The accumulation of extra cellular plaques containing the neurotoxic ß-amyloid peptide fragment Aß42 of the amyloid precursor protein APP, is one of the characteristics of Alzheimer's disease(AD). It has been shown that immunization with Aß42 reduced the development of the AD-like pathology that otherwise occurs in the transgenic mouse [3].

We found that antibodies generated during the therapeutically effective immunization with Aß42 inhibit cerebral fibrillar Aß formation, disaggregate preformed fibrils, and abrogate in vitro cell death elicited by amyloid ß peptide [4].

In this study the proteolytic reactions of APP have been probed chiefly by identification of Aß-specific epitope using new methods of mass spectrometry, particularly FT-ICR-MS as a bioanalytical tool of ultrahigh sensitivity and resolution [5].

References

1. Suckau, D., Köhl, J., Karwath, G., Scheneider, K., Bitter-Suermann, D., and Przybylski M. (1990) Molecular epitope identification by limited proteolysis of an immobilized antigen-antibody complex and mass spectrometric peptide mapping. Proc. Natl. Acad. Sci. USA. 87, 9848-9852.

2. Macht, M., Marquardt, A., Deininger, S.-O., Damoc, E., Kohlmann, M. and Przybylski, M. (2003) "Affinity-proteomics": Direct protein identification from biological material using mass spectrometric epitope mapping. Analytical and Bioanalytical Chemistry, in press.

3. Janus, C.et al., Aß peptide immunisation reduces behavioral impairment and plaques in a model of Alzheimer’s disease. Nature 408, 979-985, 2000.

4. McLaurin, J., Cecal, R., Kierstead, M. E., Tian, X., Phinney, A.L., Manea, M., French, J. E., Lambermon, M. H. L., Darabie, A. A., Brown, M. E., Janus, C., Chishti, M. A., Horne, P., Westaway, D., Fraser, P. E., Jmount, H. T. J., Przybylski, M., and St. George-Hyslop. P. (2002) Sera Generated by Aß42-vaccination Recognise a Specific Epitope, Inhibit Aß- Cytotoxiticity and Fibrillogenesis, and Induce Fibril Disaggregation. Nature Med. 8, 1263-1269.

5. Rossier, J.S., Youhnovski, N., Lion, N., Damoc, E., Becker, S., Reymond, F., Girault, H.H., Przybylski, M. (2003) A Chip-microspray System for the High Resolution FTICR Mass Spectrometry. Angew. Chem. 42/1,53-58.

On-line electrochemical tagging of free cysteine by substituted hydroquinones during nanospray ionisation for mass spectrometry in protein analysis

L. Dayon, C. Roussel, T.C. Rohner, H. Jensen and H.H. Girault*

Laboratoire d’Electrochimie Physique et Analytique,

EPFL, Lausanne, Switzerland

A nanospray interface with an integrated electrode1 coupled to a mass spectrometer has been used to electrogenerate (1,4)-benzoquinone from (1,4)-hydroquinone which reacts with free cysteine as a 1,4-Michael addition2,3. This tool has revealed itself as a very useful method to quantify cysteine in peptides or proteins and it allows to simplify proteins analysis. In order to optimise the tagging reaction coupled with MS detection different substituted hydroquinones (2-methoxyhydroquinone, 2-methylhydroquinone, methyl 2,5-dihydroxybenzoate and 2-nitrohydroquinone) were tested and compared with hydroquinone. Substituted hydroquinones were first studied in the presence of L-cysteine. The kinetic behaviours of the coupled chemical reaction were determined by cyclic voltammetry performed in the spray medium (MeOH/H2O/AcOH 50/49/1). Digital simulations were used to extract the kinetic constants and the final tagging efficiency was studied by MS using the nanospray interface. Methyl 2,5-dihydroxybenzoate gave the best results whereas 2-nitrohydroquinone was abandoned because of its inadequate tendency to polymerise. Unexpectedly, 2-methoxyhydroquinone, though it presents the smaller kinetic constant, gave good results in MS experiments because of the higher ionisation efficiency of the adduct formed from L-cysteine and 2-methoxyhydroquinone compared with the ones formed from 2-methylhydroquinone and hydroquinone. Several peptides containing up to three cysteines residues were investigated as substrates in the tagging experiment. Methyl 2,5-dihydroxybenzoate and 2-methoxyhydroquinone gave again the best tagging. The selectivity for cysteine residue was confirmed for all hydroquinones in the set-up used. b-Lactoglobuline A from bovine milk containing one cysteine residue was infused in the presence of the substituted hydroquinones through an infusion line connected to the chip. The spectrum obtained showed the native distributions of peaks for the protein and a shifted distribution corresponding to the adduct formation with the free cysteine residue.

1. T.C. Rohner, J.S. Rossier, H.H. Girault, Anal. Chem. 2001, 73, 5353-5357

2. T.C. Rohner, J.S. Rossier, H.H. Girault, Electrochem. Commun. 2002, 4, 695-700

3. C. Roussel, T.C. Rohner, H. Jensen, H.H. Girault, Chem. Phys. Chem. 2003, 4, 200-206

On-chip sample preparation for proteomics high-resolution mass spectrometry

Niels Lion1, Eugen Damoc2, Susanna Becker2, Nikolay Youhnovski2,

Michael Przybylski2 and Hubert Girault1

1 Laboratoire d’Electrochimie Physique et Analytique,

Ecole Polytechnique Fédérale de Lausanne, Switzerland

2 Laboratory of Analytical Chemistry, University of Konstanz, Germany

A microfabricated polymer-based nanospray embeding a polyvinylidene fluoride (PVDF) membrane has been designed for stop-and-go proteins and peptides desalting, which is a requisite prior to ESI-MS analysis1. Peptides and proteins are first adsorbed on the PVDF membrane through hydrophobic interactions; salts and co-solvents can then be washed out by water, and analytes subsequently eluted from the membrane and sprayed at the outlet of the microchannel with acidified methanol or acetonitrile. The use of microfluidics allows precise control of delivered volumes, and elution behaviour, so that preconcentration of the sample can be performed. This process was assessed with various concentrations of salts and co-solvents such as urea, thiourea, reducing agents2, etc. Only detergents such as CHAPS prevent retention of analytes by the membrane. Several examples of model peptide and protein analysis are shown, as well as preliminary results on the high-resolution mass spectrometric analysis of Tau protein digest.

1. Lion, N., Gobry, V., Jensen, H., Rossier, J. and Girault, H. H., Integration of a membrane based desalting in a microfabricated disposable polymer injector for mass spectrometric protein analysis, Electrophoresis, (2002) 23: 3583-3588

2. Lion, N., Gellon, J. O., Jensen, H. and Girault, H. H., On-chip protein sample desalting and preparation for direct-coupling with electrospray ionization mass spectrometry, Journal of Chromatography A, (2003) 1003: 11-19

Mapping of cellular prion protein from cerebrospinal fluid and central nervous system: immunoblotting and purification via dedicated antibodies

Annalisa Castagna1, Natascia Campostrini1, Alessia Farinazzo2, Gianluigi Zanusso2 , Salvatore Monaco2 and Pier Giorgio Righetti1

1Department of Agricultural and Industrial Biotechnologies,

University of Verona, Verona, 37134, Italy

2Department of Neurological and Visual Sciences,

University of Verona, Verona, 37134, Italy

The cellular prion protein (PrPC) is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein abundant in neurons. PrPC represents the substrate for the generation of a conformational pathogenic isoform (PrPSc) in human and animal prion diseases. By applying novel solubilization cocktails, we analyzed normal human brain and cerebrospinal fluid (CSF) PrPC by immunoblot, using specific antibodies. Here we show that PrPC from brain and CSF, is composed of several charge isomers of differently glycosylated isoforms of the full-length PrPC and some N-terminally truncated fragments. We also succeeded in capturing PrPC from brain homogenates via specific antibodies grafted to silica surfaces and are currently analysing by mass spectrometry the various isoforms eluted from 2-D maps.

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