Principles of Mass Spectrometry-Based Proteomics. Steven Gygi Department of Cell Biology Harvard Medical School, Boston, MA

Transcription

1 Principles of Mass Spectrometry-Based Proteomics Steven Gygi Department of Cell Biology Harvard Medical School, Boston, MA

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3 The Flow of Information

4 The Flow of Information Networks Complexes Proteins Genes

5 mrna Profile Patterns on a Global Level The Full Yeast Genome on a Chip Statistics: 6116 Yeast Genes 96 Intergenic regions DeRisi et al, 1997, Science 278:680

6 Control of Eukaryotic Gene Expression Nucleus Cytosol Inactive mrna DNA Trancriptional control Primary RNA transcript RNA processing control mrna RNA transport control mrna Translational control Translational control Protein Inactive protein Protein activity control Active protein

7 Proteomics: Systematic identification and characterization of proteins for their quantity, structure, function, activity and molecular interactions

8 Mass-Spectrometry-Based Proteomics Identification Intensity m/z Proteomics Intensity m/z PTMs Quantification

9 If all you have is a hammer, everything looks like a nail -- Bernard Baruch

10 Biochemists have cool hammers

11 Ion Trap Mass Spectrometry IPI:IPI Homo sapiens (Human) SPLICE ISOFORM 2 OF INSULIN-LIKE GROWTH FACTOR II PRECURSOR. MGIPMGKSMLVLLTFLAFASCCIAAYRPSETLCGGELV DTLQFVCGDRGFYFRLPGRPASRVSRRSRGIVEECCF RSCDLALLETYCATPAKSERDVSTPPTVLPDNFPRYPVG KFFQYDTWKQSTQRLRRGLPALLRARRGHVLAKELEA FREAKRHRPLIALPTQDPAHGGAPPEMASNRK

12 Unambiguously identify proteins Determine the precise site of a PTM P Ac Quantify protein abundance Intensity m/z Intensity time

13 Protein Sequencing NH 2 -Glu-Gly-Ser-Thr-Ser-Pro-Pro-His-Ala-His-Leu-Lys-COOH Edman-type degradation Tandem mass spectrometry 1 hr = Glu 1 hr = Gly 1 hr = Ser. 1 hr = Lys Total Time = 12 hours Total Time = ~1 second

14 Overview How Proteins Are Studied with Mass Spectrometry Protein/Peptide Mass Spectrometry Instrumentation Mass Spectrometry (MS) and Tandem Mass Spectrometry (MS/MS) Peptide Sequencing by MS/MS Large-Scale MS Based Proteomics Studies Large-Scale Data Acquisition Automated Assignment of MS/MS Data Studying Posttranslational Modification with MS Quantitative MS Proteomics

15 Protein/Peptide Mass Spectrometry General Scheme of a Mass Spectrometer Ionization source Mass analyzer Detector Evaporation Desorption Ionization Separation of Ions Based on their m/z (High Vacuum) Gas-phase ions

16 Protein/Peptide Mass Spectrometry Ionization of Large Biomolecules - Soft Ionization Techniques Matrix-Assisted Laser Desorption/Ionization (MALDI) Singly-Charged Ions Fast Analysis Electrospray Ionization (ESI) Multiply-Charged Ions Direct On-Line Coupling with Chromatographic Separation Techniques (LC-MS, LC-MS/MS) Nobel Prize in Chemistry 2002: John B. Fenn, Koichi Tanaka

17 Protein/Peptide Mass Spectrometry Multiply Charged Ions from the Electrospray Process Mass Determination of Large Proteins Cytochrome C (equine), molecular weight 12,360 Da Deconvoluted Spectrum Fenn et al. (1989) Science 246, 64-71

18 Protein/Peptide Mass Spectrometry Multi-Step Mass Analysis: MS and MS/MS The MS Experiment Detector Electron Multiplier ESI Quadrupole Mass Analyzer -(U + Vcosωt) z MS -(U + Vcosωt)

19 Protein/Peptide Mass Spectrometry Protein Identification at the MS level by Peptide-Mass Fingerprinting Relative High Purity of Proteins Is Required 2D gel electrophoresis Digestion with Trypsin (Peptides better Amenable to MS) >gi sp P50086 MSNYPLHQACMENEFFKVQELLHSKPSLLLQKDQDGRIPLHW SVSFQAHEITSFLLSKMENVNLDDYPDD SGWTPFHIACSVG NLEVVKSLYDRPLKPDLNKITNQGVTCLHLAVGKKWFEVSQF LIENGASVRIKDKFN QIPLHRAASVGSLKLIELLCGLGKSA VNWQDKQGWTPLFHALAEGHGDAAVLLVEKYGAEYDLVDNKG AKAEDVALNEQVKKFFLNNV MS (typically) MALDI

20 Protein/Peptide Mass Spectrometry Multi-Step Mass Analysis: MS and MS/MS The MS/MS Experiment Triple Quadrupole Analyzer Q1 Q2 Q3 Scan Rf-Only No Filter Function MS/MS Experiments Are Initialized with an MS Experiment MS

21 Protein/Peptide Mass Spectrometry Multi-Step Mass Analysis: MS and MS/MS The MS/MS Experiment Q1 Q2 Q3 Isolating Defined Ion Collision with Inert Gas (CID*) Scanning MS MS/MS 677

22 Protein/Peptide Mass Spectrometry Fragmentation of Peptide Ions x (n-m) z (n-m) R m y (n-m) H N O O R m+1 a m b m c m

23 Protein/Peptide Mass Spectrometry Fragmentation of Peptide Ions y 7 y 6 y 5 y 4 y 3 y 2 y 1 H 2 N R 1 O R 2 O R 4 N H O H N R 3 N H H N O R 5 O N H R 6 H N O R 7 O R 8 N H O OH a 2 b 2 b 3 b 4 b 5 b 6 b 7

24 Protein/Peptide Mass Spectrometry NH 2 -NSGDIVNLGSIAGR-COOH YMR134W, yeast protein involved in iron metabolism

25 Large-Scale Protein Profiling Mass Spectrometry of Peptides and Proteins

26 Large-Scale MS Based Proteomics Studies Large-Scale Protein Profiling Fractionation of Proteins High Numbers Range of Proteins in Biological Samples High Concentration Range of Proteins Limited Number of Sequence Attempts (MS/MS Experiments) in an Mass Spectrometric Analysis Limited Dynamic Range of Mass Spectrometers (Partly Due to Peptides Competing for Protons in the Ionization Process Ionization Supression)

27 Large-Scale MS Based Proteomics Studies Large-Scale Protein Profiling Why Are Peptides and Not Proteins Analyzed? Better Solubility, Allows Analysis of e.g. Membrane Proteins Better Amenable to Chromatographic Separation (Mostly RP) Better Amenable to Mass Spectrometric Analysis Ionization Fragmentation (Energy Impact in CID Process too Small to Fragment Proteins) Digestion with Trypsin (Cleaves After K and R), Protonation on both Termini of Peptide Ions b and y Ion Series Various Posttranslational Modifications (PTMs) Lead to a Combinatorial Explosion of Protein Sequence Databases

28 Large-Scale MS Based Proteomics Studies Large-Scale Protein Profiling Why Are Peptides and Not Proteins Analyzed? Intact Protein Analysis Top-Down Proteomics ( Bottom-Up for Peptides) Histone H4 (Human), Function Controlled by Cassettes of PTMs Not Accessible by Bottom-Up Proteomics Electron Capture Dissociation for Fragmentation Various Known Modifications (Acetylation, Methylation, Phosphorylation) 46,875 Sequences Considered Pesavento et al. (2003) JACS 126

29 Large-Scale MS Based Proteomics Studies Large-Scale Protein Profiling Mass Spectrometers Used in Proteomic Studies

30 Two Different Instruments with Similar Performance LTQ Orbitrap LTQ FT

31 The LTQ Orbitrap Makarov (2000) Anal. Chem. 72, Makarov (1999) US Patent 5, 886, 346.

32 The LTQ Orbitrap 1. Ions are stored in the Linear Trap 2.. are axially ejected 3.. and trapped in the C-trap 4.. they are squeezed into a small cloud and injected into the Orbitrap 5.. where they are electrostatically trapped, while rotating around the central electrode and performing axial oscillation The oscillating ions induce an image current into the two outer halves of the orbitrap, which can be detected using a differential amplifier Ions of only one mass generate a sine wave signal

33 The LTQ Orbitrap The axial oscillation frequency follows the formula ω = Where ω = oscillation frequency k = instrumental constant m/z =. well, we have seen this before k m / z Many ions in the Orbitrap generate a complex signal whose frequencies are determined using a Fourier Transformation

34 Large-Scale MS Based Proteomics Studies Large-Scale Protein Profiling Data-Dependent MS/MS Spectra Acquisition More than 10,000 MS/MS Spectra Acquired in a Typical LC-MS/MS Run

35 Large-Scale MS Based Proteomics Studies Large-Scale Protein Profiling High Mass Accuracy and High Mass Resolution: Allows Charge Determination of Peptides Enhances Confidence in Peptide Identifications Lowers the Influence of Noise in Quantitative Studies

36 Large-Scale MS Based Proteomics Studies Large-Scale Protein Profiling De Novo Sequencing of Peptides Based On MS/MS Data Ambitious Task Whole-Genome Sequence Information: Automated MS/MS Spectra Assignment Through Matching Acquired MS/MS Spectra With those Predicted on the Basis of Protein Sequence Databases Facilitates Large-Scale Proteomics

37 Large-Scale MS Based Proteomics Studies Large-Scale Protein Profiling MS/MS Spectra Database Search Algorithms: SEQUEST: Theoretical Spectra Predicted Based on Database Sequences Cross-Correlation to Match Acquired with Predicted Data Mascot: Determination of Corresponding b- and y-ions. Probability for Random Match Calculated Acquired MS/MS Spectrum In Silico Predicted Spectrum, b- and y-ions

38 Large-Scale MS Based Proteomics Studies Examples 4 Life Cycle Stages Characterized 2,400 Proteins Profiled Aim: Targets for Drugs/Vaccines to Interrupt Life Cycle Florens et al. (2002) Nature 419, 520

39 Examples Large-Scale MS Based Proteomics Studies

40 Profiling of Posttranslational Modifications Postranslational Modifications (PTM) Identifying PTMs with MS >200 known Localization, Activity State, Turnover Interaction A R N D C E Q G H I L LC-MS/MS Analogous to Analysis of Unmodified Peptides Database Search: Modified Residue(s) Considered as Additional Residue Potentially Replacing the Unmodified Residue K M F P S OR S P T OR T P W Y OR Y P V

41 Quantitative Studies by MS Stable Isotopes

42 Internal Standards S Da O NH 2 HN O O N H N H H 2 N N H O O O N H O P OH OH O OH H N M ps F E I L R O O N H O OH S Da O HN NH 2 O O N H N H H 2 N N H O O O N H O P OH OH O OH O H N O N H M ps F E I L* O OH R

43 Internal Standards in MS Stable Isotope Dilution: Enrich heavier isotope occurrence from natural abundance to ~ 100% for certain atoms in the analyte Carbon: 12 C = % 13 C = 1.07%

44 Isotope Coded Affinity Tags (ICAT) ICAT Reagents: Heavy reagent: d8-icat (X=deuterium) Light reagent: d0-icat (X=hydrogen) O N S N O N X X X O X O XX O X X N O I Biotin tag Linker (heavy or light) Thiol specific reactive group

45 The ICAT Strategy for Quantitative Proteomics

46 Principles of ICAT Strategy Quantitative Potential to identify unknown proteins Automated Complexity of peptide mixture is reduced Redundant quantification if multiple cysteines present Database search is constrained by SH-specificity Relative protein quantity is maintained through biochemical, immunological, or physical fractionation Compatible with analysis of low abundance proteins

47 Comparison of Yeast Utilizing Ethanol or Galactose as Carbon Source Galactose Transition states Ethanol 100 µg protein 100 µg protein Heavy ICAT reagent Light ICAT reagent ICAT analysis

48 Differential Protein Expression in Yeast Growing on Ethanol or Galactose Gene Name Sequence Ratio (Eth : Gal) Galrepressed Glurepressed ACH1 KHNC#LHEPHMLK >100 : 1 Y ADH1 YSGVC#HTDLHAWHGDWPLPVK 0.57 : 1 C#C#SDVFNQVVK 0.48 : 1 ADH2 YSGVC#HTDLHAWHGDWPLPTK >200 : 1 C#SSDVFNHVVK >200 : 1 Y Y PEP4 KGWTGQYTLDC#NTR 2.60 : 1 Y LPD1 VC#HAHPTLSEAFK 1.30 : 1 Y TEF1 RGNVC#GDAK C#GGIDK FVPSKPMC#VEAFSEYPPLGR 0.81 : : : 1 GAL1 LTGAGWGGC#TVHLVPGGPNGNIEK 1 : >200 Y GAL10 HHIPFYEVDLC#DR 1 : >200 DC#VTLK 1 : >200 Y PGM2 C#TGGIILTASHNPGGPENDMGIK 0.58 : 1 Y PCK1 LSIC#GEESFGTGSNHVR IPC#LADSHPK C#INLSAEKEPEIFDAIK C#AYPIDYIPSAK IVEEPTSKDEIWWGPVNKPC#SER 0.62 : : : : : 1 SOD1 GFHIHEFGDATNGC#VSAGPHFNPFK 0.46 : 1 Y QCR6 ALVHHYEEC#AER 1.30 : 1 Y Y

49 Ethanol Metabolism in Yeast Glucose or Galactose 2NAD + glycolysis Alcohol dehydrogenase isozymes 2NADH Oxaloacetate Pyruvate CH 3 -C-H Acetaldehyde TCA Cycle Oxidative Phosphorylation Acetate O 2NADH 2NAD + ADH1 ADH2 2NADH 2NAD + CH 3 -CH 2 -OH Ethanol C0 2 + H 2 0

50 ICAT Analysis of Yeast Utilizing Different Carbon Sources: Alcohol Dehydrogenase Isozymes ADH1 : YSVCHTDLHAWHGDWPLPVKADH2 : YSVCHTDLHAWHGDWPLPTK Relative Abundance Ethanol Galactose 50 Ethanol Galactose m/z m/z Ratio: 0.57 Ratio: >200

51 Metabolic Labeling For Quantitative Proteomics IR in Light DMEM [ 12 C 6, 14 N 2 ]-Lys MW: 146 [ 12 C 6, 14 N 4 ]-Arg MW: T cells in Heavy DMEM [ 13 C 6, 15 N 2 ]-Lys MW: 154 [ 13 C 6, 15 N 4 ]-Arg MW: 184 Trypsinize Enrich Phosphopeptides LC-MS/MS ID and Quantification

52 Quantification of Relative Difference in Protein Phosphorylation MS 6 Da MS 6 Da Relative Intensity Light Heavy Relative Intensity Light Heavy m/z m/z Unregulated Regulated

53 DNA Damage Induces A Phosphorylation Cascade And Cell Cycle Arrest DNA Damage Mec1 (ATR) Tel1(ATM)???? Rad53 Chk1???? Dun1??????????

54 DNA Replication Is A Dangerous Event For Complex Genomes

55 Chromosomal Breakage in RAD17 -/- Cells RAD17 +/+ RAD17 -/- Chromosome Breaks Lei Li

56 Cancer Susceptibility Linked to DNA Damage Responses Syndrome Fanconi s Anemia Werner s Syndrome Ataxia-Telangietasia Xeroderma Pigmentosa Bloom s Syndrome Nijmegen Breakage Syndrome Sekel Syndrome Li Fraumeni (p53, Chk2) HNPCC BRCA1, BRCA2 Defect Crosslink Repair/ALL Aging, Damage-sensitivity IR sensitivity, Ataxia ALL, NHL Excision Repair/Skin Cancer SCE, Leukemia IR sensitivity/ ALL, NHL Microcephaly, Checkpoint Cell cycle, Apoptosis, Many tumors Mismatch repair/colon cancer + others Recombination, Breast + Ovarian Cn

57 Measuring Phosphorylation Differences After IR Treatment

58 Quantification and identification of phospho-sq/tq peptides by LC-MS/MS MS 6 Da MS 6 Da Relative Intensity Light Heavy Relative Intensity Light Heavy m/z m/z Unregulated DNA Damage Regulated

59 Example of XIC for Quantitative Pair after DNA Damage

60 Phosphorylation Changes After IR Treatment in Yeast

61 Examples of IR-induced Phosphorylation Changes

62 Final Thoughts Mass spectrometry can identify, characterize, and quantify proteins Scale can be in the thousands not global Stable isotopes are powerful proteome-labeling atoms Large-scale experiments often require significant validation Mass spectrometry-based proteomics is enabling the measurement of new endpoints in biology

63 Gygi Lab Dept. Cell Biology Harvard Medical School Taplin Biological MS Facility Acknowledgements