Fundamentals of Biological Mass Spectrometry and Proteomics

Fundamentals of Biological Mass Spectrometry and Proteomics Steve Carr Broad Institute of MIT and Harvard Modern Mass Spectrometer (MS) Systems Orbitrap Triple Quadrupole Q-Exactive Discovery/Global Experiments Targeted MS MS systems used for proteomics have tasks: •  Create ions from analyte molecules •  Separate the ions based on charge and mass •  Detect ions and determine their mass-to-charge •  Select and fragment ions of interest to provide structural information (MS/MS) Electrospray MS: ease of coupling to liquid-based separation methods has made it the key technology in proteomics Possible Sample Inlets Syringe Pump Sample Injection Loop Autosampler, HPLC Liquid Capillary Electrophoresis Expansion of the Ion Formation and Sampling Regions Nitrogen Drying Gas Electrospray Needle Atmosphere 3-5 kV Liquid Nebulizing Gas Droplets Containing Solvated Ions Ions Vacuum Isotopes Most elements have more than one stable isotope For example, most carbon atoms have a mass of 12 Da, but in nature, 1.1% of C atoms have an extra neutron, making their mass 13 Da Why we care? Mass spectrometers “see” the isotope peaks provided the resolution is high enough If an MS instrument has resolution high enough to resolve these isotopes, better mass accuracy is achieved Stable isotopes of most abundant elements of peptides Element H C N O Mass 1.0078 2.0141 12.0000 13.0034 14.0031 15.0001 15.9949 16.9991 17.9992 Abundance 99.985% 0.015 98.89 1.11 99.64 0.36 99.76 0.04 0.20 Monoisotopic mass and isotopes We use instruments that resolve the isotopes enabling us to accurately measure the monoisotopic mass Monoisotopic mass; all 12C,mass no 13C atoms Monoisotopic corresponds to 13 One mass C atom lowest peak Two 13C atoms Angiotensin I (MW = 1295.6) (M+H)+ = C62 H90 N17 O14 TheWhen monoisotopic mass of aare molecule is the sum of thethe accurate masses for the mass most the isotopes clearly resolved monoisotopic abundant isotope of each element present As the number of atoms of any given element is used it is theofmost accurate measurement increases, theas percentage the population of molecules having one or more atoms of a heavier isotope of this element also increases The most significant contributors to the isotopic peak pattern for peptides is the 13C isotope of carbon (1.1%) and 15N peak of nitrogen (0.36%) Example of electrospray mass spectrum of mixture of peptides Isotope spacing = 1.0: Ion is singly charged: (M+H)1+ MW = 584.2 Isotope spacing = 0.5: Doubly charged: (M+2H)2+ MW = 1096.2 Isotope spacing = 0.3: Triply charged: (M+3H)3+ MW = 1806.6 586.2 603.5 Example of electrospray mass spectrum of mixture of peptides F-G-G-F-T-G-A-R-K-S-A-R-K-L-A-N-Q F-G-G-F-T-G-A-R-K-S-A F-G-G-F-T-G Nociceptin 1-11 Peptide (M +2H)2+ Peptide (-H, +Na) Peptide (M+H)1+ Peptide (M+3H)3+ 586.2 Nociceptin 1-17 Nociceptin 1-6 Peptide 3: MW = 1806.6 Peptide 2: MW = 1096.2 Peptide 1: MW = 584.2 603.5 How we sequence peptides: MS/MS Scan m/z 350-1200 Pass All ions Pass All ions MS Collision Cell (off) MS Pass m/z 834-838 Collision Cell (on) Fragment all ions Pass All ions intact peptide parent ions MS MS or mass spectrum HPLC Column MS/MS MS/MS spectrum of doubly charge ion at m/z 836.5 MS/MS means using two mass analyzers (combined in one instrument) to select an analyte (ion) from a mixture, then generate fragments from it to give structural information Dominant fragment ions observed by collisioninduced dissociation (CID) of peptides b ions Direct cleavage of peptide bond y ions Rearrangement of mobile proton SILAC: Stable Isotope Labeling by Amino acids in Cell culture Metabolic labeling (SILAC) (up to samples at a time) State A (light) State B (heavy) Label Pros Cons Deep, highly precise quant Limited plex level (3 max) Works well in most cell lines Not practical for most model systems Works with all PTMs Can’t label humans Relatively inexpensive Combine State A State B 6-7 doublings in media depleted of light (12C6)lysine m/z •  Time course of activation •  Mixing samples improves data and saves instrument time •  ID of p-sites requires MS/MS •  Detects some proteins associated with pY-proteins Blagoev, Ong et al (2004) Nature Biotech 22: 1139 Chemical Labeling of Peptides: Multiplexed Quantification with Isobaric Mass Tag Reagents Mass = 145 Chemical Labeling of Peptides: Multiplexed Quantification with Isobaric Mass Tag Reagents 100 80 116.1111 114.1108 117.1145 9060 Mix Peptides from all Samples: analyze by MS 291.2149 390.2832 Relative Abundance 8040 MS 7020 60 112 114 116 118 m/z 50 40 30 reporter ions 720.4188 503.3672 MS/MS 703.2882 404.3024 614.2397 20 116.1111 218.0594 462.1813 200.1014 331.1429 240.1341 145.1086 10 549.2076 352.1475 561.3007 100 150 200 250 300 350 400 450 500 550 600 650 m/z 792.3369 774.3190833.5016 904.5338 700 750 800 Sequence informative fragment ions 850 900 950 m/z same peptide from different samples: Observed precursor intensity = Σ of all labeled versions 116.1111 iTRAQ Experimental Example DMSO Kinase Inhib Kinase Inhib Kinase Inhib Peptide #1: No effect Relative Abundance 100 Lyse and Digest 114.1108 80 60 40 20 112 Label “116” “117” Pool Phosphopeptide Enrichment Peptide #2: Sensitive to all inhibitors Relative Abundance “115” 116 m/z 118 114.1107 100 “114” 114 80 60 115.1077 117.1146 40 20 112 LCMS 114 116 m/z 118 116.1117 100 Peptide #3: Sensitive to inhibitors & Relative Abundance 114.1112 80 60 40 117.1146 20 112 114 116 m/z 118 Isobaric tag reagents with higher multiplex levels now available: increased sample throughput with high sensitivity and good quantitative fidelity 3x increased throughput Highly consistent quantification results tumor samples (4 basal; luminal; reference) ref iTRAQ4 TMT6 TMT10 Log2 basal/luminal tumors Analytical challenges of proteomics differ in important ways from transcriptional analysis Transcriptional Profiling MS-based Proteomics Lysozyme #1047-1064 RT: 14.18-14.47 AV: 18 NL: 5.58E3 T: FTMS + p NSI Full ms2 1431.40@35.00 [ 390.00-2000.00] 993.0279 100 95 1584.7555 90 85 1167.6802 Relative Abundance 80 1408.6818 75 1352.9105 70 1538.7399 65 1638.7799 1839.5064 60 975.5341 55 50 1081.7426 45 40 1773.8836 35 1894.6001 30 25 20 1285.7981 15 559.8581 10 660.3698 1963.1184 806.9863 915.3887 497.6919 400 Ÿ All possible features known Ÿ Sample is static during analysis Ÿ All features measured Ÿ Robust means to amplify low numbers DNA or RNA (PCR) Ÿ Signal not detected means feature not present 600 800 1000 1200 m/z 1400 1600 1800 2000 Ÿ All possible features not known Ÿ Sample is dynamic during analysis Ÿ 20-50% of features measured Ÿ No protein PCR (analytics have to deal with enormous dynamic range) Ÿ Signal not detected means either that feature not present or feature present but not detected Discovery defines a reduced set of “sentinel” marks that need to be repeatedly measured in a range perturbations Discovery: • Disease • Development • Drug • KO/KI Not all proteins and (especially) PTMs observed in all experiments Currently: Westerns, immunoassays Future: Targeted MS, ImmunoMS Assays: • Highly specific • Sensitive • Highly precise • Multiplexed • Interference-free Precisely measure selected analytes in all experiments – no missing data How you start a proteomics project? We meet to discuss your project (scarr@broadinstitute.org) •  Project proposals are reviewed for scientific merit, technical feasibility and alignment with our interests and the Broad mission •  Discussion of the science and experimental design •  Sample preparation discussed in detail - what, how and by whom •  All projects are collaborative Funding: •  Platforms are largely self-supporting and must charge the work performed If projects are reviewed favorably but lack funding, we will help investigators explore options for support, including consideration for collaborative funding through the Broad www.broadins$tute.org/proteomics   The Broad Institute Proteomics Group Suggested additional reading Carr and Annan, 2001 Overview of Peptide and Protein Analysis by Mass Spectrometry Current Protocols in Molecular Biology 10: 10.21.1–10.21.27 Aebersold, R and Mann, M 2003 Mass spectrometry-based proteomics Nature 422:198-207 Cravatt et al 2007 The biological impact of mass-spectrometry-based proteomics Nature 450: 991-1000 Additional resources for MS data interpretation •  Manual  de  novo  tutorials     •  Don  Hunt  and  Jeff  Shabanowitz     •  h]p://www.ionsource.com/tutorial/DeNovo/DeNovoTOC.htm   •  Rich  Johnson   •  h]p://www.abrf.org/ResearchGroups/MassSpectrometry/EPosters/ms97quiz/SequencingTutorial.html   •  Automated  de  novo  -­‐  PEAKS   •  h]p://www.bioinformaHcssoluHons.com/peaks/tutorials/denovo.html   •  h]p://www.youtube.com/watch?v=lyhpRu6s7Ro   •  De  Novo  Sequencing  and  Homology  Searching  Tutorial   •  Ma  B,  Johnson  R  Mol  Cell  Proteomics  11: O111.014902,  1–16,  2012   •  ModificaHon  Site  LocalizaHon  Scoring:  Strategies  and  Performance  -­‐  Review   •  Chalkley,  RJ  and  Clauser,  KR  Mol  Cell  Proteomics  11,  3-­‐14,  2012     •  Target/Decoy  FDR  -­‐  Tutorial   •  Elias  &  Gygi,    Nature  Methods,  4,  207-­‐214,  2007   •  Protein  Inference    -­‐  Tutorial   •  Nesvizhskii,    Mol  Cell  Proteomics,  4,  1419-­‐1440,  2005   41 BACKUPS   14+ 13+ ESI MS 23,984 +/- Example of electrospray mass spectrum (M+H)1+ = 24,010 +/- 30 of intact protein (beta-Casein) MALDI MS 24,024 +/- Relative Abundance, % + 18+ 15 16+ 24,093 +/- 12+ 1400 1410 1420 11+ 10+ 9+ 8+ 800 1600 1200 m/z 2000 20000 22000 24000 m/z 26000 47 M Mann, C K Meng and J B Fenn, Anal Chem 61, 1702 (1989) ... 10.21.1–10.21.27 Aebersold, R and Mann, M 2003 Mass spectrometry- based proteomics Nature 422:198-207 Cravatt et al 2007 The biological impact of mass- spectrometry- based proteomics Nature 450: 991-1000... complex biological samples –  Data interpretation is far more difficult and less automated –  Breadth and depth of coverage of the proteome is orders of magnitude less than for bottom-up proteomics. .. www.broadins$tute.org /proteomics   The Broad Institute Proteomics Group Suggested additional reading Carr and Annan, 2001 Overview of Peptide and Protein Analysis by Mass Spectrometry Current