Martin.4.09

Introduction to Mass spectrometry Based Proteomics Workflow

What is proteomics?

Proteomics includes not only the identification and quantification of proteins, but also the determination of their localization, modifications, interactions, activities, and, ultimately, their function.

-Stan Fields in Science, 2001.

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Dynamic Range =103

How to think about mass spectrometry based proteomics

mRNAAble to amplify (PCR)

Protein No amplification

High variability in amount (>109)

How to think about mass spectrometry based proteomics

What it is: A highly powerful tool for protein identification and quantification Complementary to other technologies and analysis methods What it is not: Magic Able to give all the answers Simple (relatively speaking) Cheap

Mass Spectrometry based proteomics: What it is and what it isnt

What can mass spectrometry tell me?

proteins in mixtures quantitative analysis of protein expression post-translational modifications:

Phosphorylation

protein interactions

Mass Spectrometry PrimerA mass spectrometer

measures mass to charge ratio or m/z

Ionization Mass Separation Ion collection

quadrupole ion trap time-of-flight

MALDI Electrospray mass analysis

Protein, MW = 10,000 +

digest into peptides cleaves C-teminal side of arginine (R) and lysine (K)

TrypsinN-THK.NCPHIVVGTPGR.IPD-C

What do we put in our mass spectrometer to measure m/z?

NH2NH2

NH2

-COOHR

K-COOH

-COOHK

Peptides, MW < 4,000

+

+

+

+

+

+

High voltage placed on a fused silica column causes a spray of charged droplets which evaporate leaving charged peptides

NewObjective

UniversityofBristol

NH2 K-COOH++

Electrospray ionization (ESI)

Matrix-assisted laser desorption/ionization (MALDI)

3 mm

matrix sampleCOOH

OH

CH CNC

UV laser

A laser pulse excites the matrix material at its resonant frequency and energy is imparted to peptides, charging them.

Triple Quadrupole Mass SpectrometerTriple Quadrupole Mass Spectrometer

electrospray sourceelectrospray sourceIonizationIonization

Q1Q1 Q2Q2 Q3Q3

collision cellcollision cellMass SelectionMass Selection

DetectorDetectorIon CollectionIon Collection

microcapillarymicrocapillary

Quadrupole Optics

In a quadrupole mass spectrometer four (quad) parallel rods (poles) are arranged equidistantly from a central (imaginary) axis.

Charged ions are injected along the central axis of the quadrupole assembly.

Static and alternating (radio frequency) electric potentials are applied to opposite pairs of rods, creating a fluctuating electric field.

NN

N

N

N

Mass Spectrometers identify peptides by fragmenting along the amide backbone

++NSGDIVNLGSIAGRNSGDIVNLGSIAGR++

b yb y

++N SGDIVNLGSIAGRN SGDIVNLGSIAGR++++NS GDIVNLGSIAGRNS GDIVNLGSIAGR++++NSG DIVNLGSIAGRNSG DIVNLGSIAGR++++NSGD IVNLGSIAGRNSGD IVNLGSIAGR++++NSGDIV NLGSIAGRNSGDIV NLGSIAGR++++NSGDIVN LGSIAGRNSGDIVN LGSIAGR++++NSGDIVNL GSIAGRNSGDIVNL GSIAGR++++NSGDIVNLG SIAGRNSGDIVNLG SIAGR++++NSGDIVNLGS IAGRNSGDIVNLGS IAGR++++NSGDIVNLGSIA GRNSGDIVNLGSIA GR++++NSGDIVNLGSIAG RNSGDIVNLGSIAG R++

Fragmentation occurs along the backbone, revisited

Y ions

++NSGDIV NLGSIAGRNLGSIAGR++

RRGGIISS AAGGLLNN

++NN+ + NSNS+ + NSGNSG+ + NSGDNSGD+ + NSGDI NSGDI + + NSGDIVNSGDIV+ + NSGDIVNNSGDIVN+ + NSGDIVNLNSGDIVNL+ + NSGDIVNLGNSGDIVNLG+ + NSGDIVNLGSNSGDIVNLGS+ + NSGDIVNLGSINSGDIVNLGSI+ + NSGDIVNLGSIANSGDIVNLGSIA+ + NSGDIVNLGSIAGNSGDIVNLGSIAG+ + RR+ + RGRG+ + RGARGA+ + RGAIRGAI+ + RGAISRGAIS+ + RGAISGRGAISG+ + RGAISGLRGAISGL+ + RGAISGLN RGAISGLN + + RGAISGLNVRGAISGLNV+ + RGAISGLNVIRGAISGLNVI+ + RGAISGLNVIDRGAISGLNVID+ + RGAISGLNVIDGRGAISGLNVIDG+ + RGAISGLNVIDGSRGAISGLNVIDGS

++NSGDIVNLGSIAGRNSGDIVNLGSIAGR++

bions

bions

yions

yions

The fragment ladder allows sequence assignment

200200 400400 600600 800800 10001000 12001200

m/zm/z

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HH22 NN --NSGDIVNLGSIAGRNSGDIVNLGSIAGR--COOHCOOH

GGDD

II

VVNN

LLGGSS

IIAAGGRR

NN SSGG

DDII

VV NN LLGG

Quadrupole Optics cont.

The quadrupole can function in a second mode called tandem The quadrupole can function in a second mode called tandem mass spectrometry or MS/MS.mass spectrometry or MS/MS.

A particular peak is chosen from a MS scan and the first quad A particular peak is chosen from a MS scan and the first quad allows only that m/z to pass into the second quad.allows only that m/z to pass into the second quad.

The second quad accelerates the species through a voltage The second quad accelerates the species through a voltage causing collisions with an inert gas present.causing collisions with an inert gas present.

If the ion is a peptide, the collisions cause bond breakage If the ion is a peptide, the collisions cause bond breakage selectively at the selectively at the amideamide bondsbonds

The charged fragments enter the third quad which performs a MS The charged fragments enter the third quad which performs a MS scan generating a unique pattern associated with the fragments scan generating a unique pattern associated with the fragments and thus the parent peptideand thus the parent peptide

These fragments can be deconvoluted to give a peptide sequenceThese fragments can be deconvoluted to give a peptide sequence

Ar

Ar600600 800800 10001000 12001200 14001400

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peptides

trypsin

gel

Tandem mass spectrometry

ESIESI

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tandem mass spectrumtandem mass spectrum

fragment peptide

Ar

Ar

ArAr

1. MS survey scan

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Sample PeptideSample Peptide

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HH22 NN --NSGDIVNLGSIAGRNSGDIVNLGSIAGR--COOHCOOH

GGDD

II

VVNN

LLGGSS

IIAAGGRR

NN SSGG

DDII

VV NN LLGG

Making an identification by database searching using SEQUEST

SEQUEST is a search program that assigns a peptide sequence to a spectra by comparing it to virtual spectra from a protein database

1.1. An MS/MS scan of m/z 750 and charge 2+An MS/MS scan of m/z 750 and charge 2+ the the molecular weight is 1500 Damolecular weight is 1500 Da

2.2. SEQUEST searches a protein database starting at the SEQUEST searches a protein database starting at the first amino acid to find all possible peptides that weight first amino acid to find all possible peptides that weight 1500 +/1500 +/-- 1.51.5

3.3. SEQUEST fragments each virtually and compares to the SEQUEST fragments each virtually and compares to the experimental spectra. experimental spectra.

4.4. For a good spectra, one peptide stands out from all For a good spectra, one peptide stands out from all othersothers

SEQUEST ExampleSEQUEST Example

Scoring a match

4 5 6 7 83210 9 10 11

4 5 6 7 83210 9 10 11

4 5 6 7 83210 9 10 11

4 5 6 7 83210 9 10 11

4 5 6 7 83210 9 10 11

4 5 6 7 83210 9 10 11

NSGDIVNLGSIAGR NSADIVNLGSIAGR

Acquired spectra

Theoretical spectra

Dotproduct

The linear ion trap LTQ

The Orbitrap and LTQ-FT

Average vs. monoisotopic mass

LTQ-FT

Time of Flight

All ions are imparted with the same energy (E) by the application of an electric field.

E=mv2/2

m=2E/v2

Distance/time=v

m=2E/(d/t)2

Mass can be calculated based on time of arrival because the energy, flight tube length, and time are

known.

Scanning with a ESI-tof

High mass accuracy Low duty cycle Large data files

ESIESI

Electron Transfer Dissociation

PNAS February 13, 2007 vol. 104 no. 7 21932198

C

ETD

Source: Thermo Fisher

Quantitative Proteomics

Why?...Comparisons

Particularly well suited to answering biologic questions

Detail protein level changes to a biologic stimulus Used as a tool to evaluate protein distributions Used as a tool to follow complex formation

Modified from CEBI web site

Isotopic modification strategies

Mass spectrometers measure mass-hence isotopically different peptides can be compared.

Single ion chromatograms for each isotope can be compared to determine relative quantities of each.

550550 560560 570570 580580

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lightlight heavyheavy

The general formats of isotope addition

Metabolic labeling Tagging of Cysteine Tagging of free amines 18O labeling during digestion Spiking in a heavy peptide to quantify a target

Isotopic tags

Label should react with a group common in proteins

Labeling must be complete Labeling must be stable Label cannot inhibit ionization or fragmentation Label must provide a large enough mass

difference for peaks to be resolved. Light and heavy peptides should co-elute in all

separation steps preceding mass spectrometry

Stable Isotope Labeling with Amino acids in Cell culture

SILAC

Cells are grown in media lacking Arginine or Lysine

These amino acids are supplied as either heavy (13C6 ,

15N4 Arginine and 13C6 Lysine) or light into media Serum is dialyzed to remove amino

acids

SILAC

Heavy Lysine,

Arginine or both

Unmodified amino acids

+

Media w/o Lys and Arg

FCS dialyzed to remove amino acids

Stimulus of

interest

Control

Lyse cells together

SILAC

Very simple, grow cells and lyse together. Dialyzed serum may cause growth issues in some

cell lines All peptides are labeled- this can challenge the

mass spec duty cycle in some experiments Not applicable to cells that cannot be easily

propagated i.e. primary cells Not applicable to samples where propagation is

not possible i.e. mammalian serum or organs

Protein Labeling

Isotopic tagging at the protein level is most commonly done through cysteine or lysine modification.

A variety of reactants are available that incorporate thiol specific chemistry with variable isotopic tags. Some are selective

Amine based reagents are available as well.

Cleavable Isotope Coded Affinity Tags

Heavy reagent: 13CHeavy reagent: 13C--ICAT ICAT Light Light reagentreagent: 12C: 12C--ICATICAT

S

N N

O

SH

cys?I

NO

ON

O

NPeptide

O

ON

OS

H2 N

Acid cleavage

Protein Quantification and Identification by the ICAT StrategyProtein Quantification and Identification by the ICAT Strategy

Affinity Affinity separation separation with Avidinwith Avidin

Combine and Combine and proteolyzeproteolyze

ICATICAT-- labeled labeled

cysteinescysteines

Mixture 2Mixture 2

Mixture 1Mixture 1

ProteinsProteinsPeptidesPeptides

ICAT

Selective and therefore reduces complexity potentially increasing dynamic range

Most proteins have a cysteineHowever:

Method involves a variety of handling steps that can cause sample loss

Parallel cell lysis and handling can introduce bias.

Peptide level labeling

amine based prototypes N-isotag iTRAQ

Can be amine or cysteine based

iTRAQ

iTRAQ (isotope Tags for Relative and Absolute Quantitation).

Commercially available from ABI Requires CID to generate reporter ions for

quantitation. Requires a TOF instrument

NH2

NH2

NH2

NH2

iTRAQ schema

CID of iTRAQ labeled peptides

Copyright 2004 American Society for Biochemistry and Molecular Biology

Ross, P. L. (2004) Mol. Cell. Proteomics 3: 1154-1169

ITRAQ

Copyright 2004 American Society for Biochemistry and Molecular Biology

Ross, P. L. (2004) Mol. Cell. Proteomics 3: 1154-1169

Labelling is consistent within one protein

iTRAQ summary

Very effective method for multiplexed quantitative studies

Trades off increased ID/quantification using many peptides vs. selection strategy (ICAT)

Is not compatible with LC/MS strategies that choose CID peaks based on pair intensity ratios

Label Free Quantitative Proteomics

Isotopes are not used Quantification is performed computationally by comparing

sequential runs Metrics of comparison are typically AUC for aligned runs MS/MS is either incorporated or done later as targeted

MS/MS As an alternative, peptide counting can be used as a method

for relative quantification with MS/MS experiments.

How is this technology implemented?

Large scale

Small scale

Medium scale

Big project example

4000 proteins

>300 orbitrap runs

Big group

Big $$$$$$

Medium Scale Proteomics

The key to medium scale proteomics is a mechanical or affinity based preparation that reduces the complexity of the starting material.

Examples: centrifugation, precipitation.

Peroxisome membrane

We have combined classical subcellular fractionation with large-scale quantitative mass spectrometry to identify proteins that enrich specifically with peroxisomes of Saccharomyces cerevisiae.

Micro-Proteomics

Focus on a particular protein complex Affinity based purification Takes advantage of existing technology Results more approachable for an individual

investigator

Micro-Proteomics

Yknown

unknown

Release

Identification of binding partners

Conclusions 1

Proteomics is rapidly advancing: Relative quantification is here. Large scale experiments are becoming easier with

better automation tools, BUT they generate vast amounts of data and consume significant resources.

Medium and small scale projects can be approached by an individual investigator here and now.

Phosphorylation is observable but methodology is still under development.

Conclusions 2 Mass spectrometers are fantastic

The results you get out are determined by what you put in.

The results you get out are determined by what you put in

The results you get out are determined by what you put in.

Problems are most often NOT the result of poor instrument performance

Your results depend on the following: The purity and cleanliness of you preparation The complexity of your sample compared to the dynamic range and duty cycle of the instrument

Introduction to Mass spectrometry Based Proteomics Workflow What is proteomics?How to think about mass spectrometry based proteomicsHow to think about mass spectrometry based proteomicsMass Spectrometry based proteomics: What it is and what it isntWhat can mass spectrometry tell me?Mass Spectrometry PrimerWhat do we put in our mass spectrometer to measure m/z?Electrospray ionization (ESI)Slide Number 10Slide Number 11Quadrupole OpticsSlide Number 13Slide Number 14Mass Spectrometers identify peptides by fragmenting along the amide backbone Fragmentation occurs along the backbone, revisitedY ionsSlide Number 18The fragment ladder allows sequence assignmentQuadrupole Optics cont.Slide Number 21Slide Number 22Making an identification by database searching using SEQUESTSlide Number 24Scoring a matchThe linear ion trap LTQThe Orbitrap and LTQ-FTSlide Number 28Average vs. monoisotopic massLTQ-FTSlide Number 31Time of FlightSlide Number 33Scanning with a ESI-tofSlide Number 35Electron TransferDissociationETDETDETDQuantitative ProteomicsWhy?...ComparisonsSlide Number 42Isotopic modification strategiesThe general formats of isotope additionIsotopic tagsStable Isotope Labeling with Amino acids in Cell cultureSILACSILACSILACProtein LabelingCleavable Isotope Coded Affinity TagsSlide Number 51ICATPeptide level labelingiTRAQiTRAQ schemaCID of iTRAQ labeled peptidesSlide Number 57Slide Number 58Slide Number 59iTRAQ summaryLabel Free Quantitative Proteomics How is this technology implemented?Slide Number 63Big project exampleMedium Scale ProteomicsPeroxisome membraneMicro-ProteomicsMicro-ProteomicsConclusions 1Conclusions 2

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Martin.4.09