Peptide profiling by capillary separation techniques coupled to mass
spectrometry
Gaspari, Marco
Citation
Gaspari, M. (2006, December 13). Peptide profiling by capillary separation techniques coupled to mass spectrometry. Retrieved from https://hdl.handle.net/1887/5431
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in theInstitutional Repository of the University of Leiden Downloaded from: https://hdl.handle.net/1887/5431
Peptide Profiling by Capillary Separation Techniques
Coupled to Mass Spectrometry
Proefschrift ter verkrijging van
de graad van Doctor aan de Universiteit van Leiden, op gezag van de Rector Magnificus Dr. D.D. Breimer,
hoogleraar in de faculteit der Wiskunde en Natuurwetenschappen en die der Geneeskunde, volgens besluit van het College voor Promoties
te verdedigen op woensdag 13 december 2006 te klokke 13.45 uur
door
Marco Gaspari geboren te Cosenza, Italië
Promotiecommissie
Promotor: Prof.dr. J. van der Greef
Co-promotor: Dr. E. Verheij
Referent: Prof.dr. H. Irth
Overige leden: Prof.dr. M. Danhof
Prof.dr. T. Hankemeier
Prof.dr. R. Verpoorte
Printed by Grafisch bedrijf Ponsen & Looijen B. V., Wageningen, The Netherlands.
ISBN 9074538665
Contents
Chapter 1 Capillary chromatographic separations and mass spectrometry 9
1.1 Mass spectrometry 9
1.1.1 Electrospray ionisation 9 1.1.2 Nanoelectrospray ionisation 11
1.1.3 Mass analysis 14
1.2 Capillary chromatographic separations 17 1.2.1 Nanoscale liquid chromatography 18 1.2.2 Capillary electrochromatography 19 1.3 Interfacing separations by nanoES ionisation 21
Chapter 2 Proteomics and peptide profiling 27 2.1 Two-dimensional gel electrophoresis 28
2.1.1 Protein identification by mass spectrometry 30 2.2 Bottom-up LC-MS proteomics 32 2.2.1 Single-dimensional nanoLC-MS approaches 32 2.2.2 Multi-dimensional nanoLC-MS approaches 33 2.2.3 Quantitative bottom-up proteomics 36 2.3 2DE versus bottom-up LC-MS 38
2.3.1 Key parameters 39
2.3.2 Experimental evidences 43 2.3.3 Final considerations 44
2.4 Data analysis 46
2.4.1 Data analysis in MS-based proteomics 46
2.4.2 Multivariate analysis 48
2.4.3 Multivariate analysis in proteomics 50
Scope of the thesis 51
Chapter 3 55
3.1 Capillary electrochromatography/ nanoelectrospray 57 mass spectrometry for attomole characterization of peptides
Chapter 4 Fabrication of narrow-bore packed capillary columns 85 for high-efficiency nanoscale LC-MS analysis of peptide
mixtures
Chapter 5 Novel Strategies in Mass Spectrometric Data Handling 101
Chapter 6 115
6.1 Protein transacetylase of buffalo liver: characterization 117 and mass spectrometry of the acetylated protein product
6.2 Discovery of modified peptides in protein tryptic digests by 135 automated handling of capillary LC-MS data
Chapter 7 Integration of two-dimensional LC-MS with multivariate 143 statistics for comparative analysis of proteomic samples
Chapter 8 167
Conclusions and perspectives 169
Summary 171
Samenvatting 173
Curriculum Vitae 175
Acknowledgement 177
