ebi is an outstation of the european molecular biology laboratory. introduction to mass spectrometry...

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



Overview …

• Basics of Mass Spectrometry

• Proteomics approaches: bottom-up/shot-gun

• Fractionation and depletion techniques

• MS modes

• Instrument sep-ups available



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



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).



MASS SPECTROMETRY:

CONCEPTS AND COMPONENTS



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.



MS PROTEOMICSWORKFLOW



MS proteomics: Shot-gun/bottom-up approaches

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100

%

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%

MS analysis

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100

%

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%

MS/MS analysis

fragmentation

PROTOCOL

peptides

proteins

sequencedatabase



1. SAMPLE (PROTEIN) FRACTIONATION



Protein Fractionation: Gel electrophoresis

1D SDS gel

MW MW

pI

2D SDS gel

2D gel image from: http://www.fixingproteomics.org/

It



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



2. PROTEIN DIGESTION




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MS analysis

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%

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MS/MS analysis

fragmentation

PROTOCOL

peptides

proteins

sequencedatabase



A

A

I

K

G

K

I

D

VC

I

V

L

L

Q H KA

E PT

I

R

NT

DG

R

TA

Start with a protein



Cut with a protease (trypsin)A

A

I

K

G

K

I

D

VC

I

V

L

L

Q H KA

E PT

I

R

NT

DG

R

TA



Select a peptideA

A

I

K

G

K

I

D

VC

I

V

L

L

Q H KA

E PT

I

R

NT

DG

R

TA



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



Digest with trypsinMAAKDVKFGRTAREKMLRGVDILADAVKVTLGPKGRNVVI EKSFGAPRITKDGVSVAKEVELEDKFENMGAQMLRVQTSKPLLIIAEDVEGEALATLVVNKEVASKTNDTAGDGTT TATVLGQAIVQEGAKAVAAG MNPMDLKGI DLAVNEVVAELLKKAINT SEEVAQVGTI SANGEAEIGKMIAEAMQKVG NEGVITVEEA KTAETELEVVEGMQFDRGYLSPYFVTNPEKMVADLEDAYILLHEKLSNLQALLPVLEAVLR

GGLKIAAVKAPGFGDCRAMLEDIAILTGGQV ISEDLGIKLESVTLDMLGRAKVSISKENTTIVDGAGQKAEIDARVGQIKQQIEETTSDYDREKLQERLAKLAGGVAVIRVGGATEVEVKDRVDDALNATRAAVEEGIVAGGGTALL RASTKITAKGVNADQEAGIN IVRAIQAPARQITTNAGEEASVIVGKILENTSETFGYNTANGEYGDLISLGIVDPVKVVRTALQNAASVAGLLITTEAMIAELPKDAAPAGMPGGMGGMGGMDF



3. PEPTIDE SEPARATION (CHROMATOGRAPHY)




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%

MS analysis

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100

%

100 300 500 700 900 1100 1300 1500 1700 1900 2100m/z0

100

%

MS/MS analysis

fragmentation

PROTOCOL

peptides

proteins

sequencedatabase



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.



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).



4. MASS SPECTROMETRY




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%

MS analysis

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100

%

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MS/MS analysis

fragmentation

PROTOCOL

peptides

proteins

sequencedatabase



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



MS MODES



MS modes

• PMF: Peptide Mass Fingerprinting

• MS/MS: Tandem MS

• MRM/SRM (Multiple/Selected Reaction Monitoring)



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)



TANDEM MASS SPECTROMETRY

(TANDEM-MS, MS/MS, MS2)



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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



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?



MS proteomics: MS2 spectra



Fragmentation techniques

• PSD: Post-Source Decay

• CID: Collision Induced Dissociation

• ETD/ ECD (Electron Transfer Dissociation/ Electron Capture Dissociation)



Mass Spec Principles

Ionization Source

Sample

+_

Mass Analyzer/s Detector




Ionization Source- MALDI- ESI

Sample

+_





Ionization Source

Sample

+_

Mass Analyzer/s

-Time of Flight (TOF)- Ion Trap (IT)- Quadrupole (Q)- FTICR- Orbitrap

Detector




Ionization Source

Sample

+_


- Electron multiplier



Comparison between the instruments

From: Domon & Aebersold, Science, 2006



A SPECIAL FLAVOUR OF MS/MS:

MULTIPLE/SELECTED REACTION MONITORING



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)



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



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



Questions?

ebi is an outstation of the european molecular biology laboratory. introduction to mass spectrometry...

Documents

ebi roadshow rotterdam

ms proteomics workflow

ebi proteomes uniprot

pride protein families

mass spectrometry ms

ups available slide

protein expression biomarkers

cambridge united kingdom