High Accuracy Mass Spectrometric Peptide Identification As a Discovery Tool in Proteomics
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Dissertation zur Erlangung des Doktorgrades Der Fakultät für Chemie und Pharmazie Der Ludwig-Maximilians-Universität München High accuracy mass spectrometric peptide identification as a discovery tool in proteomics Alexandre Zougman Aus Odessa, Ukraine 2008 2009 Erklärung Diese Dissertation wurde im Sinne von § Abs. 3 der Promotionsordnung vom 29. Januar 1998 von Herrn Prof. Dr. Matthias Mann betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet. München, am February 19, 2009 ...................................................... (Unterschrift des Autors) Dissertation eingereicht am February 19, 2009 1. Gutachter Prof. Dr. Matthias Mann 2. Gutachter Prof. Dr. Jacek R. Wiśniewski Mündliche Prüfung am March 23, 2009 Contents Abbreviations .............................................................................................................................. 3 Summary ..................................................................................................................................... 4 Introduction ................................................................................................................................. 5 Mass spectrometry ......................................................................................................... 5 Generating ions .............................................................................................................. 7 MALDI .......................................................................................................................... 8 ESI ................................................................................................................................. 9 Assessing the limits of protein detection ..................................................................... 11 Isotopic envelopes ....................................................................................................... 12 Accuracy and resolution .............................................................................................. 14 Analyzing the ionized peptides ................................................................................... 15 Tryptic peptides and fragmentation ............................................................................. 17 NanoLC-MS ................................................................................................................ 18 LC-MS/MS and protein database searches ................................................................. 23 The Q-TOF .................................................................................................................. 28 The Orbitrap ................................................................................................................ 31 HCD ............................................................................................................................. 35 De novo sequencing as a proteomic tool ..................................................................... 37 Proteomics in genomics ............................................................................................... 37 Neuropeptidomics ....................................................................................................... 38 Presented work .......................................................................................................................... 40 Proteomics as a tool for discovery of novel genetic editing mechanisms ....................... 40 Evidence for insertional RNA editing in humans ....................................................... 44 Future directions .................................................................................................... 65 CSF proteome and peptidome profiling ........................................................................... 66 Integrated analysis of the CSF peptidome and proteome ............................................ 69 Future directions .................................................................................................... 87 Outlook ..................................................................................................................................... 88 References ................................................................................................................................. 89 Acknowledgements ................................................................................................................... 91 Curriculum vitae ....................................................................................................................... 92 Abbreviations amu: atomic mass unit CID: collision induced dissociation CNS: central nervous system CSF: cerebrospinal fluid EST: expressed sequence tags ESI: electrospray ionization ET: extended protein FT: Fourier transform FT-ICR: Fourier transform ion cyclotron resonance FWHM: full width of the peak at half of its maximum height GC: gas chromatography HCD: high energy C-trap dissociation HPLC: high performance liquid chromatography ICR: ion cyclotron resonance LC-MS: liquid chromatography mass spectrometry LTQ: linear trap quadrupole m/z: mass-over-charge ratio MALDI: matrix-assisted laser desorption ionisation MS: mass spectrometry MS/MS: tandem mass spectrometry fragmentation NanoLC: nanoflow liquid chromatography NanoES: nanoelectrospray ppm: part per million PTM: post-translational modification Q-TOF: quadrupole time of flight RF: radio frequency RP: reverse phase RT: retention time TOF: time of flight UV: ultraviolet light wavelength 3 Summary The recent achievements of proteomics are mainly due to the developments in the high accuracy and resolution mass spectrometry. Protein identification by conventional proteomics “shotgun” approach involves enzymatic cleavage and consequent identification of the resulting peptide products. Typically, several peptides belonging to the same unique protein are produced by this process and, considering the improved sensitivity and separation abilities of the contemporary LC-MS instrumentation, it is feasible to identify a known protein with at least two unique peptides by submitting a query to a protein database. In order to be time efficient, a typical LC- MS run includes high accuracy identification of the precursor peptide mass during the MS scan event and significantly less accurate but fast MS/MS peptide fragmentation signature profiling. This scheme generally satisfies very stringent protein identification requirements. However, identification of endogenous peptides such as neuropeptides, for example, often must rely on one peptide only and, hence, has to be much more accurate in order to provide the needed level of confidence. Likewise, the MS/MS data have to be very accurate for identification and characterization of novel post-translational modifications. Furthermore, de-novo sequencing of unknown proteins not present in protein databases frequently relies initially on the discovery of only one peptide which provides the basis for the following characterization of the unknown protein. For the above-mentioned cases the availability of high accuracy data in both MS and MS/MS modes is of the utmost importance. In my work I utilized high accuracy mass spectrometry for discovery of novel extended forms of nuclear proteins and underlying mRNA editing events, and, also, for characterization of the human cerebrospinal fluid peptidome and proteome. 4 “Tout progrès scientific est un progrès de method.” “All scientific progress is progress of a method.” René Descartes Introduction The extraordinary achievements of current proteomics are based largely on successful developments in the fields of mass spectrometry (MS) and separation science. Once joined, the two disciplines provided a powerful tool to investigate the protein universe. Mass spectrometry Mass spectrometry is the science of determining mass-to-charge ratios (m/z) of ionized molecules. In 1897 Sir Joseph John Thomson, a British physicist, in a series of experiments with cathode rays discovered the existence of the electron and determined its mass to charge ratio m/z (1). He was awarded the Nobel Prize in physics in 1906 and is considered a founder of the field of mass spectrometry. It took more than half a century of research and development before a first commercial mass spectrometer appeared, and almost a century to fully (or almost fully) seize the analytical potential of the MS instruments. This development has gone in many directions – with different instrument designs using different physical principles. However, the essence of any mass spectrometer is the same – to determine the m/z of an introduced ion. William Stephens of the University of Pennsylvania presented the idea of the time-of-flight (TOF) mass analyzer at the 1946 American Physical Society Meeting in Cambridge. His concept was to accelerate all ions to the same kinetic energy. Thus an accelerated ion would have the same kinetic energy as any other ion (provided they had the same charge), with the velocity depending solely on m/z (2). Wiley and McLaren of Bendix Corporation in Detroit introduced a TOF focusing scheme that improved mass resolution by correcting energy distributions of the ions (3). As the result, Bendix was the first to commercialize TOF instruments in the 1950s. In 1973 Boris Mamyrin, a 5 Russian scientist who worked at the Leningrad Physico-Technical Institute, described the reflectron and the energy compensating mirror system which