Complementary Application of ESI and MALDI Based Mass Spectrometry in Microbial Proteomics
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Complementary application of ESI and MALDI based mass spectrometry in microbial proteomics Inauguraldissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) an der mathematisch naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald vorgelegt von Bernd Heßling geboren am 24.08.1980 in Bocholt Greifswald, September 2012 Dekan: Prof. Dr. Klaus Fesser 1. Gutachter: Prof. Dr. Michael Hecker 2. Gutachter: Prof. Dr. Andreas Tholey Tag der Promotion: 07.01.2013 Table of Contents List of abbreviations and symbols ............................................................................................................. I Scope and outline of this thesis ............................................................................................................... II Articles and author contribution ............................................................................................................. IV 1 Introduction ........................................................................................................................... 1 1.1 Technical requirements for mass spectrometry-driven proteomics ........................................ 1 1.2 The two prevalent proteomic workflows utilizing mass spectrometry .................................... 3 2 The history of mass spectrometry driven microbial proteomics (article I) ............................... 6 3 LC-ESI-MS for global proteomics and post-translational modification analysis ........................ 9 3.1 Global proteome studies (article II) ........................................................................................ 9 3.2 Investigation of post-translational modification (article III and IV) ........................................ 13 4 Proteomics for systems biology ............................................................................................ 14 4.1 Dynamic adaptations of B. subtilis metabolism analyzed in a systems biology approach (article V) ............................................................................................................................. 15 4.2 A newly developed approach to obtain absolute protein quantities (article VI) .................... 16 5 Exchangeability and complementarity of ESI-MS and MALDI-MS .......................................... 19 5.1 Global relative quantification with LC-MALDI-MS (article VII) ............................................... 20 5.2 Complementary of MALDI and ESI (article VII) ...................................................................... 22 6 Conclusion ........................................................................................................................... 23 7 References ........................................................................................................................... 25 Article I .................................................................................................................................................. 31 Article II ................................................................................................................................................. 43 Article III ................................................................................................................................................ 91 Article IV ............................................................................................................................................... 99 Article V .............................................................................................................................................. 123 Article VI ............................................................................................................................................. 131 Article VII ............................................................................................................................................ 141 Affirmation .......................................................................................................................................... 177 Curicculum vitae .................................................................................................................................. 178 Acknowledgement .............................................................................................................................. 179 List of abbreviations and symbols 1D one dimensional MS mass spectrometry 2D two dimensional MS/MS tandem mass spectrometry B. subtilis Bacillus subtilis MSF membrane shaving fraction; BEF biotinylation-enriched fraction OD optical density ChIP chromatin immunoprecipitation PAGE polyacrylamide gel electrophoresis DNA deoxyribonucleic acid pH pondus hydrogenii e.g. for example pI isoelectric point EMF enriched membrane protein fraction PMF peptide mass fingerprimt ESI electrospray ionization PTM post-translational modification FTICR Fourier-transform ion cyclotron Q quadrupole resonance QconCAT quantification concatemer GFP green fluorescent protein RNA ribonucleic acid HPLC high-performance liquid S. aureus Staphylococcus aureus chromatography SDS sodium dodecyl sulfate i.e. that is SILAC stable isotope labeling by/with IT ion trap amino acids in cell culture ITRAQ isobaric tags for relative and absolute quantitation TOF time of flight VISA vancomycin intermediate LC liquid chromatography Staphylococcus aureus MALDI matrix assisted laser VRSA vancomycin resistant desorption/ionization Staphylococcus aureus MRSA methicillin resistant Furthermore, the usual codes for amino acids Staphylococcus aureus and chemical elements were used. Page I Scope and outline of this thesis Scope and outline of this thesis This thesis will discuss the different fields of application of the two soft ionization techniques ESI and MALDI in microbial proteomics and their importance for a better understanding of bacteria physiology. The general development in the past 25 years coming from 2D-gel analysis and protein identification by peptide mass fingerprint analysis via MALDI-TOF to genome wide quantitative LC-ESI-MS experiments with fast and sensitive ESI instruments is exemplary shown for the Gram-positive bacterium Bacillus subtilis in article I. Even though 2D-PAGE in conjunction with MALDI-MS is still an important tool in proteomic research, the more recently established global quantitative LC-ESI-MS workflows gain more and more relevance as they overcome 2D-PAGE based protein restrictions and enable the acquisition of higher accurate protein quantities. In article II such a workflow was used to analyze the physiological adaptation of Staphylococcus aureus to vancomycin treatment on a global-scale. Also post-translational modifications of proteins, that are important for regulation of their activity and allow rapid adaption to changed environmental conditions, could be analyzed by LC-ESI-MS workflows using special enrichment strategies ( article III and IV ). Despite the mentioned discrimination and less accurate quantification of proteins, 2D-PAGE analyses are still advantageous when analyzing large-scale time series experiments. To gain highly time resolved data but also very accurate relative quantities on a global-scale, 2D-PAGE-MALDI-MS and LC-ESI-MS techniques have been combined to investigate dynamic proteome adaptations of B. subtilis during nutrition shift as part of a global systems biology approach (article V ). Also absolute quantities of proteins are of high interest for systems biology, but are still challenging to obtain on large-scale as well as with sufficient accuracy. In article VI a method that again combined 2D- PAGE-MALDI-MS and LC-ESI-MS was introduced to gain absolute protein quantities on global-scale. Utilizing the complementarity of 2D-PAGE and LC-ESI-MS this new workflow enabled fast and cost efficient data acquisition on absolute scale. In article VII we described for the first time a global quantitative LC-MALDI-MS workflow. Cross validation with an LTQ Orbitrap proofed that LC-MALDI-MS is able to process complex samples and obtain highly reliable quantities. The comparative analysis of data gained with both instrument types Page II Scope and outline of this thesis revealed biases for certain biochemical properties of MALDI as well as ESI instruments, resulting in a general complementarity of both ionization techniques. Page III Scope and outline of this thesis Articles and author contribution Article I Becher, D., Büttner, K., Moche, M., Hessling, B., Hecker, M., 2011. From the genome sequence to the protein inventory of Bacillus subtilis . Proteomics 11, 2971–2980. B. H. assisted in writing the manuscript. Article II Hessling, B., Bonn, F., Herbst, F.-A., Rappen, G.-M., Bernhardt, J., Hecker, M. and Becher, D. Global proteome analysis of vancomycin stress in Staphylococcus aureus . Submitted to Mol. Cell Proteomics . B.H., F.-A. H., F.B. and G.-M.-R. performed the experiments; B.H. analyzed the data; B.H. wrote the manuscript. Article III Elsholz, A.K.W., Turgay, K., Michalik, S., Hessling, B., Gronau, K., Oertel, D., Mäder, U., Bernhardt, J., Becher, D., Hecker, M., Gerth, U., 2012. Global impact of protein arginine phosphorylation on the physiology of Bacillus subtilis . Proc. Natl. Acad. Sci. U.S.A . 109, 7451–7456. B.H. and K.G. performed the mass spectrometry analysis. Article IV Chi, B.K., Gronau, K., Mäder, U., Hessling, B., Becher, D., Antelmann, H., 2011. S- bacillithiolation protects against hypochlorite stress in Bacillus subtilis