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2. Introduction to Shot Gun Proteomics

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2. Introduction to Shot Gun Proteomics MDC m/z mass spectrometry Introduction to mass spectrometry Gunnar Dittmar What is mass spectrometry? What is the accuracy of a mass spectrometer? MDC m/z mass spectrometry Accuracy: 0.1u = 1,6 * 10-28 kg Examples: Hydrogen atom: 1u Oxygen atom: 16 u Neutron: 1 u Electron: 0.005 u MDC Uses for mass spectrometry m/z mass spectrometry Identification of chemicals quality control biological degradation Water quality control Control of food and beverages check for pesticides, toxins etc Detection of explosives toxic chemicals Doping MDC Mass spectrometry general setup m/z mass spectrometry Samples Mass Ionization Detector analyzer Data analysis MDC General principle of mass spectrometry m/z mass spectrometry + + + + + + + + + + + + electrostatic field + + + + + + + + - - - - - - - - - - - - - - - - ion source mass analyzer detector MDC General principle of mass spectrometry m/z mass spectrometry + + + + + + + + + + + + + + + + + + + + + + + + - - - - - - - - - - - - - - - - + ion source mass analyzer detector MDC Mass spectrometry m/z mass spectrometry Sir Joseph John Thomson, Nobel prize in physics for the discovery of the electron, 1906 MDC Mass spectrometry m/z mass spectrometry Sir Francis Aston, first fully functional mass spectrometer in 1919 MDC m/z mass spectrometry Ionization MDC Ionization m/z mass spectrometry ESI soft ionization Proteomics Electro-spray ionization MALDI Matrix assisted laser Proteomics desorption ionization FACS-MS hard ionization Plasma CyTOF MDC MALDI m/z mass spectrometry Image from Ekman et al. Mass spectrometry MDC MALDI m/z mass spectrometry • laser is used • sample is crystallized with a matrix • mostly singly charged ions • pulsed method • rather resistant to salt MDC Ionization m/z mass spectrometry Image from Ekman et al. Mass spectrometry ESI 2000 - 3000 V 150°C MASS nanoflow needle SPECTROMETER ESI MASS SPECTROMETER + + PFASGHFK + + + + + PFASGHFK + PFASGHFK + PFASGHFK + + PFASGHFK + PFASGHFK PFASGHFK PFASGHFK + + + + + PFASGHFK + MDC m/z mass spectrometry Mass spectrometers MDC Mass spectrometers in proteomics m/z mass spectrometry • Ion traps • LTQ • OrbiTrap • FT-ICR • TOF (time of flight) • QQQ (triple quadrupols) MDC Mass spectrometers in proteomics m/z mass spectrometry • Ion traps • LTQ • OrbiTrap • FT-ICR • TOF (time of flight) • QQQ (triple quadrupols) MDC m/z mass spectrometry Quadrupols MDC m/z mass spectrometry + - - + MDC m/z mass spectrometry - + + - MDC m/z mass spectrometry - RF RF + + - MDC m/z mass spectrometry - + + - MDC Quadrupol m/z mass spectrometry Quadrupoles of the AB Sciex Q-TRAP 5500 MDC m/z mass spectrometry Orbitrap MDC Orbitrap m/z mass spectrometry r! z! φ Korsunskii M.I., Basakutsa V.A. Sov. Physics-Tech. Phys. 1958; 3: 1396. Knight R.D. Appl.Phys.Lett. 1981, 38: 221. Gall L.N.,Golikov Y.K.,Aleksandrov M.L.,Pechalina Y.E.,Holin N.A. SU Pat. 1247973, 1986. MDC m/z mass spectrometry MDC Orbitraps m/z mass spectrometry • high mass accuracy • relatively fast MDC m/z mass spectrometry Mass spectrometers for proteomics Hybrid mass spectrometers MDC m/z mass spectrometry MDC m/z mass spectrometry proteomics shot-gun on an OrbiTrap MDC Bottom-up/Top-down m/z mass spectrometry Bottom-up peptides Top-down MDC Top-down ms m/z mass spectrometry Liquid-chromatography coupled mass spectrometry MDC m/z mass spectrometry I t MDC MS Spectrum m/z mass spectrometry MDC Top5 identification cycle m/z mass spectrometry MDC Mass spectrometry of peptides m/z mass spectrometry Ionization + PFASGHFK PFASGHFK Mass Analyzer Mass Detector + TSSSGHR TSSSGHR + HLFWTK HLFWTK m/z MDC Mass measurement of a peptide m/z mass spectrometry • exact mass of a peptide • no sequence information MDC Tandem MS or MS/MS m/z mass spectrometry PFASGHFK TSSSGHR Mass Analyzer Mass Detector HLFWTK m/z Selection of a peptide Ion Fragmentation Cell PF PFA PFAS P PFASG SGHFK ASGHFK GHFK HFK PFASGHFK FASGHFK Mass Detector m/z How to get the sequence information from Mass MDC Spectra? m/z mass spectrometry PFASGHFK PFASGHF PFASG PFAS PF P m/z PFASGHFK FASGHFK ASGHFK GHFK HFK FK K MDC Top5 cycling between MS and MS/MS m/z mass spectrometry MS MS/MS of the 1. peak MS/MS of the 2. peak MS/MS of the 3. peak MS/MS of the 4. peak MS/MS of the 5. peak MS MS/MS of the 1. peak MS/MS of the 2. peak MDC MS - MS/MS m/z mass spectrometry MDC m/z mass spectrometry Proteomes What is actually a proteome? MDC Proteome m/z mass spectrometry • “genome“ refers to all genes in a given organism • the term “proteome“ was coined by Marc Wilkins 1994 to describe the protein complement of the genome • refers to all proteins present is a sample (organism, cell, body fluid etc.) • but how can we identify all proteins? • classical approach: two-dimensional gel electrophoresis MDC 2D-gel based proteomics m/z mass spectrometry Is this a complete proteome? MDC Gel based proteomics m/z mass spectrometry Coomassie and silver staining can only detect the most abundant proteins: not sensitive enough for complete proteome analysis! MDC Protein abundance in the proteome m/z mass spectrometry Protein Copy number Serum albumine 1E+10 Transcription factors 10 - 1E5 MDC Dynamic range m/z mass spectrometry Mount Everest: 8850 m difference 1e10 bacterium: 1 µm MDC chormatography coupled proteomics m/z mass spectrometry ! Intensity !me! ESI$ MS Intensity m/z proteins peptides Database search MS/MS Intensity m/z MDC Protease digest m/z mass spectrometry MDC m/z mass spectrometry MDC chormatography coupled proteomics m/z mass spectrometry ! Intensity !me! ESI$ MS Intensity m/z proteins peptides Database search MS/MS Intensity m/z Reversed phase high performance liquid MDC chromatography (rpHPLC) m/z mass spectrometry C18 pumpA mobile phases mixer analyzer stationary phase pumpB Buffer A: water + HAc Buffer B: organic solvent + HAc Reversed phase high performance liquid MDC chromatography (rpHPLC) m/z mass spectrometry mixed analytes mobile phase (low amount of organic solvent) stationary phase (C18) Reversed phase high performance liquid MDC chromatography (rpHPLC) m/z mass spectrometry adsorption (hydrophobic interaction with C18 chains) Reversed phase high performance liquid MDC chromatography (rpHPLC) m/z mass spectrometry higher amount of organic solvent desorption Reversed phase high performance liquid MDC chromatography (rpHPLC) m/z mass spectrometry high amount of organic solvent separated analytes MDC Nano-flow HPLC m/z mass spectrometry smaller column -> lower flow rate -> higher concentration of peptides -> higher sensitivity Ideal: 20 nl/min in practise: 200 nl/min (0.2 µl/min) Nano HPLC coupled to an LTQ-OrbiTrap mass spectrometer MDC m/z mass spectrometry MDC m/z mass spectrometry Sensitivity Mass resolution MDC chormatography coupled proteomics m/z mass spectrometry ! Intensity !me! ESI$ MS Intensity m/z proteins peptides Database search MS/MS Intensity m/z Critical parameters for mass spectrometers MDC m/z mass spectrometry • Sensitivit •ability to detect small amounts of peptides • Dynamic range •ability to detect peptides with big differences in abundance • Speed •ability to fragment many peptides per second • Resolution •ability to differentiate peptides with similar m/z MDC Resolution in mass spectrometry m/z mass spectrometry m/z MDC Average mass and monoisotopic mass m/z mass spectrometry Resolution 1,000 Resolution 10,000 centroid = average mass Average m/z = 923.93 monoisotopic m/z = 923.40 z = 2 Mass of singly charged peptide ([M+H]+) = 923.40 x 2 – 1.008 = 1845.80 peptide: AEGWNFQDEHGEDRR MDC The importance of high resolution m/z mass spectrometry peptide mixture: AEGWNFQDEHGEDRR ([M+H]+ = 1845.79) VSAYVKPMITHALPYR ([M+H]+ = 1846.05) R = 1,000 MDC The importance of high resolution m/z mass spectrometry peptide mixture: AEGWNFQDEHGEDRR ([M+H]+ = 1845.79) VSAYVKPMITHALPYR ([M+H]+ = 1846.05) R = 5,000 MDC The importance of high resolution m/z mass spectrometry peptide mixture: AEGWNFQDEHGEDRR ([M+H]+ = 1845.79) VSAYVKPMITHALPYR ([M+H]+ = 1846.05) R = 10,000 MDC The importance of high resolution m/z mass spectrometry peptide mixture: AEGWNFQDEHGEDRR ([M+H]+ = 1845.79) VSAYVKPMITHALPYR ([M+H]+ = 1846.05) R = 60,000 -18 MDC Sensitivity: atto molar (10 ) m/z mass spectrometry atto molar MDC Dynamic range of an LTQ-Orbitrap m/z mass spectrometry Mount Everest: 8850 m 5000 100 000 human: petri dish: 1.8 m 10 cm LTQ-Orbitrap Q-Exactive MDC m/z mass spectrometry Data interpretation MDC Interpretation of MS/MS spectra m/z mass spectrometry PFASGHFK PFASGHF PFASG PFAS PF P m/z PFASGHFK FASGHFK ASGHFK GHFK HFK FK K MDC Automatic data analysis m/z mass spectrometry We know: • mass of precursor peptide (from MS scan) • masses of fragments (from MS/MS scan) • enzyme we have used • organism we are analyzing MDC Automatic data analysis m/z mass spectrometry 1. organism Database with all human proteins 2. enzyme in silico digest All theoretical human peptides Select peptides matching 3. mass of precursor to precursor mass (high mass accuracy important!) candidate peptides compare theoretical fragment masses 4. masses of fragments with observed fragment masses score 96 identified peptide score 5 score 2 From peptides to proteins: the protein inference MDC problem m/z mass spectrometry William of Ockham, 1288-1348: Ockhams razor principle: entia non sunt multiplicanda praeter necessitatem (entities should not be multiplied beyond necessity) Ø Report the smallest list of proteins that is sufficient to explain all identified peptides Nesvizhskii and Aebersold, 2005 Controlling the false-positive rate in shotgun MDC proteomic data m/z mass spectrometry • every
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