EBI is an Outstation of the European Molecular Biology Laboratory.
Introduction to Mass Spectrometry
Dr. Juan Antonio VIZCAINO
PRIDE Group coordinatorPRIDE team, Proteomics Services Group
PANDA group
European Bioinformatics Institute
Hinxton, Cambridge
United Kingdom
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
Databases: proteomics resources at the EBI
ProteomesUniProt, PRIDE
ProteomesUniProt, PRIDE
Protein families, motifs and domains
InterPro
Protein families, motifs and domains
InterPro
Protein structurePDBe
Protein structurePDBe
Protein interactionsIntAct
Protein interactionsIntAct
PathwaysReactome
PathwaysReactome
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
Overview …
• Basics of Mass Spectrometry
• Proteomics approaches: bottom-up/shot-gun
• Fractionation and depletion techniques
• MS modes
• Instrument sep-ups available
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
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
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
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).
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
MASS SPECTROMETRY:
CONCEPTS AND COMPONENTS
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
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.
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
MS proteomics: Shot-gun/bottom-up approaches
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MS analysis
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MS/MS analysis
fragmentation
PROTOCOL
peptides
proteins
sequencedatabase
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
1. SAMPLE (PROTEIN) FRACTIONATION
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
Protein Fractionation: Gel electrophoresis
1D SDS gel
MW MW
pI
2D SDS gel
2D gel image from: http://www.fixingproteomics.org/
It
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
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
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
MS proteomics: Shot-gun/bottom-up approaches
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MS analysis
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MS/MS analysis
fragmentation
PROTOCOL
peptides
proteins
sequencedatabase
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
A
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Q H KA
E PT
I
R
NT
DG
R
TA
Start with a protein
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
Cut with a protease (trypsin)A
A
I
K
G
K
I
D
VC
I
V
L
L
Q H KA
E PT
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NT
DG
R
TA
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
Select a peptideA
A
I
K
G
K
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D
VC
I
V
L
L
Q H KA
E PT
I
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NT
DG
R
TA
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
Digest with trypsin
>RBME00320 Contig0311_1089618_1091255 EC-mopA 60 KDa chaperonin GroEL
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
546 aa 60 kDa; 57 461 Da pI = 4.75
The sequence of the generated peptides is known
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
Digest with trypsinMAAKDVKFGRTAREKMLRGVDILADAVKVTLGPKGRNVVI EKSFGAPRITKDGVSVAKEVELEDKFENMGAQMLRVQTSKPLLIIAEDVEGEALATLVVNKEVASKTNDTAGDGTT TATVLGQAIVQEGAKAVAAG MNPMDLKGI DLAVNEVVAELLKKAINT SEEVAQVGTI SANGEAEIGKMIAEAMQKVG NEGVITVEEA KTAETELEVVEGMQFDRGYLSPYFVTNPEKMVADLEDAYILLHEKLSNLQALLPVLEAVLR
GGLKIAAVKAPGFGDCRAMLEDIAILTGGQV ISEDLGIKLESVTLDMLGRAKVSISKENTTIVDGAGQKAEIDARVGQIKQQIEETTSDYDREKLQERLAKLAGGVAVIRVGGATEVEVKDRVDDALNATRAAVEEGIVAGGGTALL RASTKITAKGVNADQEAGIN IVRAIQAPARQITTNAGEEASVIVGKILENTSETFGYNTANGEYGDLISLGIVDPVKVVRTALQNAASVAGLLITTEAMIAELPKDAAPAGMPGGMGGMGGMDF
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
3. PEPTIDE SEPARATION (CHROMATOGRAPHY)
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
MS proteomics: Shot-gun/bottom-up approaches
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MS analysis
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MS/MS analysis
fragmentation
PROTOCOL
peptides
proteins
sequencedatabase
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
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 Cation eXchange). Charge.
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
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).
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
MS proteomics: Shot-gun/bottom-up approaches
300 400 500 600 700 800 900 1000 1100m/z0
100
%
300 400 500 600 700 800 900 1000 1100m/z0
100
%
MS analysis
100 300 500 700 900 1100 1300 1500 1700 1900 2100m/z0
100
%
100 300 500 700 900 1100 1300 1500 1700 1900 2100m/z0
100
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MS/MS analysis
fragmentation
PROTOCOL
peptides
proteins
sequencedatabase
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
sample ion source mass analyzer(s) detector digitizer
Generalized mass spectrometer
- All mass analyzers operate on gas-phase ions using electromagnetic fields. The lattercan be in absolute or relative measurements.- The ion source therefore makes sure that (part of) the sample molecules are ionizedand brought into the gas phase. - The detector is responsible for actually recording thepresence of ions. Time-of-flight analyzers also require a digitizer (ADC).
Schematic view of a generalized mass spec
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
MS modes
• PMF: Peptide Mass Fingerprinting
• MS/MS: Tandem MS
• MRM/SRM (Multiple/Selected Reaction Monitoring)
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
Peptide Mass Fingerprinting (MS)
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MS analysis
Peptide MassFingerprinting
(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 forGel Based approaches
(in this case the Molecular Weight of the protein is known)
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
TANDEM MASS SPECTROMETRY
(TANDEM-MS, MS/MS, MS2)
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
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MS analysis
Peptide MassFingerprinting
(PMF)
MS/MS
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Peptide sequence information
(on top of Mass and Charge)
Fragmentation
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
NH2 CH
CO COOHNH
CH2R1
R2
CH
CO NH
CH
CO NH
CH
CH2
R3
R4
x3 y3 z3 x2 y2 z2 x1 y1 z1
a1 b1 c1 a2 b2 c2 a3 b3 c3
peptide structure
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.
Why tandem-MS?
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
Fragmentation techniques
• PSD: Post-Source Decay
• CID: Collision Induced Dissociation
• ETD/ ECD (Electron Transfer Dissociation/ Electron Capture Dissociation)
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
Mass Spec Principles
Ionization Source
Sample
+_
Mass Analyzer/s Detector
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
Mass Spec Principles
Ionization Source- MALDI- ESI
Sample
+_
Mass Analyzer/s Detector
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
Mass Spec Principles
Ionization Source
Sample
+_
Mass Analyzer/s
-Time of Flight (TOF)- Ion Trap (IT)- Quadrupole (Q)- FTICR- Orbitrap
Detector
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
Mass Spec Principles
Ionization Source
Sample
+_
Mass Analyzer/s Detector
- Electron multiplier
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
Comparison between the instruments
From: Domon & Aebersold, Science, 2006
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
A SPECIAL FLAVOUR OF MS/MS:
MULTIPLE/SELECTED REACTION MONITORING
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
mass filter 1 mass filter 2collisioncell
selectedpeptides
fragmentsof both
peptides
selectedfragment
MRM/SRM removes noise, yielding better signal-to-noise ratioMRM/SRM removes ‘contaminating’ peaks, aiding targeted identification
MRM/SRM works well with proteotypic peptidesMRM/SRM can be performed with Q-Q-Q, Q-LIT and IT instruments
peptidemixture
Multiple/Selected Reaction Monitoring (MRM/SRM)
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
31 minESI Q3
961.5
m/z
collision
m/z
SRM transitioncomplementC3= 606.4 / 961.5 at RT 31 min
SRM = selected reaction monitoring
IHWESASLLR
Q1
606.4
m/z
EBI RoadshowRotterdam, 12 June 2012
Juan A. Vizcaí[email protected]
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