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Introduction to Mass Spectrometry

Introduction to Mass Spectrometry. Dr. Juan Antonio VIZCAINO PRIDE Group coordinator. PRIDE team, Proteomics Services Group PANDA group European Bioinformatics Institute Hinxton , Cambridge United Kingdom. Databases: proteomics resources at the EBI. Protein families,

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Introduction to Mass Spectrometry

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  1. Introduction to Mass Spectrometry Dr. Juan Antonio VIZCAINO PRIDE Group coordinator PRIDE team, Proteomics Services Group PANDA group European Bioinformatics Institute Hinxton, Cambridge United Kingdom

  2. Databases: proteomics resources at the EBI Protein families, motifs and domains InterPro Protein structure PDBe Protein interactions IntAct Pathways Reactome Proteomes UniProt, PRIDE

  3. Overview … • Basics of Mass Spectrometry • Proteomics approaches: bottom-up/shot-gun • Fractionation and depletion techniques • MS modes • Instrument sep-ups available

  4. Genome vs. Proteome • Genome • Essentially static over time • Non location specific • Human genome mapped (2000) • ~20,000 genes • PCR is available to amplify DNA • Proteome • Dynamic over time • Location specific • Human proteome non-mapped: • ~400,000 proteins??? • No equivalent of PCR for proteins

  5. Exclusive information through MS proteomics • Sometimes there is not much correlation between gene expression and protein expression… • Biomarkers: easy access to human fluids (plasma, urine, …) • Post-Translational Modifications (PTMs).

  6. MASS SPECTROMETRY: CONCEPTS AND COMPONENTS

  7. Mass Spectrometry MS is an analytical technique that measures the mass-to-charge (m/z) ratio of charged particles. It is used for determining masses of particles, for the determination of the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and other chemical compounds. Results are therefore plotted on a cartesian system with mass-over-charge (m/z) on the X axis and ion intensity on the Y-axis.

  8. MS PROTEOMICS WORKFLOW

  9. MS proteomics: Shot-gun/bottom-up approaches MS/MS analysis P R O T O C O L peptides sequence database proteins fragmentation MS analysis

  10. 1. SAMPLE (PROTEIN) FRACTIONATION

  11. Protein Fractionation: Gel electrophoresis MW It pI MW 1D SDS gel 2D SDS gel 2D gel image from: http://www.fixingproteomics.org/

  12. Protein Fractionation 2: Chromatography • Way to deplete the sample. Discard the most abundant proteins in the sample to be able to ‘see’ the less abundant ones. • In complex samples, only a small proportion of peptides can be detected. • Affinity Chromatography in different flavours • Often used in plasma studies

  13. 2. PROTEIN DIGESTION

  14. MS proteomics: Shot-gun/bottom-up approaches MS/MS analysis P R O T O C O L peptides sequence database proteins fragmentation MS analysis

  15. A Start with a protein A I E P A T H K K Q I G L R L K N V I T I D D C G V R T A

  16. A Cut with a protease (trypsin) A I E P A T H K K Q I G L R L K N V I T I D D C G V R T A

  17. A Select a peptide A I E P A T H K K Q I G L R L K N V I T I D D C G V R T A

  18. Digest withtrypsin 546 aa 60 kDa; 57 461 DapI = 4.75 >RBME00320 Contig0311_1089618_1091255 EC-mopA 60 KDachaperoninGroEL MAAKDVKFGR TAREKMLRGV DILADAVKVT LGPKGRNVVI EKSFGAPRIT KDGVSVAKEV ELEDKFENMG AQMLREVASK TNDTAGDGTT TATVLGQAIV QEGAKAVAAG MNPMDLKRGI DLAVNEVVAE LLKKAKKINT SEEVAQVGTI SANGEAEIGK MIAEAMQKVG NEGVITVEEA KTAETELEVV EGMQFDRGYL SPYFVTNPEK MVADLEDAYI LLHEKKLSNL QALLPVLEAV VQTSKPLLII AEDVEGEALA TLVVNKLRGG LKIAAVKAPG FGDCRKAMLE DIAILTGGQV ISEDLGIKLE SVTLDMLGRA KKVSISKENT TIVDGAGQKA EIDARVGQIK QQIEETTSDY DREKLQERLA KLAGGVAVIR VGGATEVEVK EKKDRVDDAL NATRAAVEEG IVAGGGTALL RASTKITAKG VNADQEAGIN IVRRAIQAPA RQITTNAGEE ASVIVGKILE NTSETFGYNT ANGEYGDLIS LGIVDPVKVV RTALQNAASV AGLLITTEAM IAELPKKDAA PAGMPGGMGG MGGMDF • The sequence of the generated peptides is known

  19. Digest withtrypsin GGLK IAAVK APGFGDCR AMLEDIAILTGGQV ISEDLGIK LESVTLDMLGR AK VSISK ENTTIVDGAGQK AEIDAR VGQIK QQIEETTSDYDR EK LQER LAK LAGGVAVIR VGGATEVEVK DR VDDALNATR AAVEEGIVAGGGTALL R ASTK ITAK GVNADQEAGIN IVR AIQAPAR QITTNAGEEASVIVGK ILENTSETFGYNTANGEYGDLISLGIVDPVK VVR TALQNAASVAGLLITTEAMIAELPK DAAPAGMPGGMGGMGGMDF MAAK DVK FGR TAR EK MLR GVDILADAVK VTLGPK GR NVVI EK SFGAPR ITK DGVSVAK EVELEDK FENMGAQMLR VQTSKPLLIIAEDVEGEALATLVVNK EVASK TNDTAGDGTT TATVLGQAIVQEGAK AVAAG MNPMDLK GI DLAVNEVVAELLK KA INT SEEVAQVGTI SANGEAEIGK MIAEAMQK VG NEGVITVEEA KTAETELEVVEGMQFDR GYLSPYFVTNPEK MVADLEDAYILLHEK LSNLQALLPVLEAVLR

  20. 3. PEPTIDE SEPARATION (CHROMATOGRAPHY)

  21. MS proteomics: Shot-gun/bottom-up approaches MS/MS analysis P R O T O C O L peptides sequence database proteins fragmentation MS analysis

  22. Sample Fractionation: Peptide separation • One technology that has been key in the development of proteomics is the concurrent miniaturization and automation of liquid chromatography • In complex samples, only a small proportion of peptides can be detected. • Two main types of chromatography are used for peptides: • - Reverse-Phase electrophoresis. Hydrophobicity. • - SCX (Strong CationeXchange). Charge.

  23. Sample Fractionation: Peptide separation • Chromatography and MS are ‘on-line’ for ESI approaches. This is not possible for MALDI. • ‘Retention Time’ is an essential piece of information to be taken into account (another dimension to be added to m/z).

  24. 4. MASS SPECTROMETRY

  25. MS proteomics: Shot-gun/bottom-up approaches MS/MS analysis P R O T O C O L peptides sequence database proteins fragmentation MS analysis

  26. Schematic view of a generalized mass spec sample ion source mass analyzer(s) detector digitizer Generalized mass spectrometer • All mass analyzersoperate on gas-phase ions using electromagnetic fields. The latter • can be in absolute or relative measurements. • - The ion source therefore makes sure that (part of) the sample moleculesare ionized • and brought into the gas phase. • - The detector is responsible for actually recording the • presence of ions. Time-of-flight analyzers also require a digitizer (ADC).

  27. MS MODES

  28. MS modes • PMF: Peptide Mass Fingerprinting • MS/MS: Tandem MS • MRM/SRM (Multiple/Selected Reaction Monitoring)

  29. Peptide Mass Fingerprinting (MS) MS analysis Peptide Mass Fingerprinting (PMF) MW - Each peak in the spectrum represents a peptide (or mixture of peptides) - Information about the Mass and Charge Not very used at present except for Gel Based approaches (in this case the Molecular Weight of the protein is known)

  30. TANDEM MASS SPECTROMETRY (TANDEM-MS, MS/MS, MS2)

  31. MS/MS MS analysis Peptide Mass Fingerprinting (PMF) Fragmentation Peptide sequence information (on top of Mass and Charge) MS/MS analysis

  32. Why tandem-MS? peptide structure x3 y3 y2 z2 y1 z3 x2 x1 z1 R2 R3 R1 CH2 CH2 R4 NH2 C H CO N H C H CO N H C H CO N H C H COOH a1 b1 c1 a2 b2 c2 a3 b3 c3 There are several other ion types that can be annotated, as well as ‘internal fragments’. The latter are fragments that no longer contain an intact terminus. These are harder to use for ‘ladder sequencing’, but can still be interpreted. This nomenclature was coined by Roepstorff and Fohlmann(Biomed. Mass Spec., 1984) and Klaus Biemann (Biomed. Environ. Mass Spec., 1988) and is commonly referred to as ‘Biemann nomenclature’. Note the link with the Roman alphabet.

  33. MS proteomics: MS2 spectra

  34. Fragmentation techniques • PSD: Post-Source Decay • CID: Collision Induced Dissociation • ETD/ ECD (Electron Transfer Dissociation/ Electron Capture Dissociation)

  35. Mass Spec Principles Sample + _ Detector Ionization Source Mass Analyzer/s

  36. Mass Spec Principles Sample + _ Detector Ionization Source - MALDI - ESI Mass Analyzer/s

  37. Mass Spec Principles Sample + _ Detector Ionization Source • Mass Analyzer/s • -Time of Flight (TOF) • Ion Trap (IT) • Quadrupole (Q) • FTICR • Orbitrap

  38. Mass Spec Principles Sample + _ Detector - Electron multiplier Ionization Source Mass Analyzer/s

  39. Comparison between the instruments From: Domon & Aebersold, Science, 2006

  40. A SPECIAL FLAVOUR OF MS/MS: MULTIPLE/SELECTED REACTION MONITORING

  41. Multiple/Selected Reaction Monitoring (MRM/SRM) collision cell mass filter 1 mass filter 2 peptide mixture selected peptides fragments of both peptides selected fragment MRM/SRM removes noise, yielding better signal-to-noise ratio MRM/SRM removes ‘contaminating’ peaks, aiding targeted identification MRM/SRM works well with proteotypicpeptides MRM/SRM can be performed with Q-Q-Q, Q-LIT and IT instruments

  42. SRM = selected reaction monitoring Q1 collision Q3 ESI 31 min m/z m/z m/z 606.4 961.5 IHWESASLLR SRM transitioncomplementC3= 606.4 / 961.5 at RT 31 min

  43. Conclusions • Important concepts leant: bottom-up, top-down proteomics • Not all the peptides can be ‘seen’ by the instrumentation • Workflows used to decrease the complexity of the sample • PMF and MS/MS, also MRM/SRM • Different instrument set ups

  44. Questions?

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