Peptide Sequencing by Peptide Sequencing by

Mass SpectrometryMass Spectrometry

Alex RamosAlex Ramos

5 April 20055 April 2005

EdmanEdman degradationdegradation

N C S H 2 N C

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A sp P he P he A rg C

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Pe ptide s ho rthe ne d by o ne re s idue

P he nyl iso th io c ya na te

EdmanEdman Degradation v. MS/MSDegradation v. MS/MS

Why study proteins?Why study proteins?

�� machines that make cells functionmachines that make cells function

�� RNA levels do not always accurately predict RNA levels do not always accurately predict

protein levelsprotein levels

�� targets of drugstargets of drugs

Peptide AnalysisPeptide Analysis

�� EdmanEdman DegradationDegradation

�� MSMS

�� More sensitiveMore sensitive

�� Can fragment peptides fasterCan fragment peptides faster

�� Does not require proteins or peptides to be purified to Does not require proteins or peptides to be purified to

homogeneityhomogeneity

�� Has no problem identifying blocked or modified proteinsHas no problem identifying blocked or modified proteins

IntroductionIntroduction

�� MS/MS plays important role in protein identification (fast MS/MS plays important role in protein identification (fast and sensitive)and sensitive)

�� Derivation of peptide sequence an important task in Derivation of peptide sequence an important task in proteomicsproteomics

�� Derivation without help from a protein database (Derivation without help from a protein database (““de novo de novo sequencingsequencing””), especially important in identification of ), especially important in identification of unknown proteinunknown protein

Basic lab experimental stepsBasic lab experimental steps1. 1. Proteins digested w/ an enzyme to produce peptidesProteins digested w/ an enzyme to produce peptides

2. Peptides charged (ionized) and separated according 2. Peptides charged (ionized) and separated according

to their different to their different m/zm/z ratiosratios

3. Each peptide fragmented into ions and 3. Each peptide fragmented into ions and m/zm/z values of values of

fragment ions are measuredfragment ions are measured

�� Steps 2 and 3 performed within a tandem mass Steps 2 and 3 performed within a tandem mass

spectrometer.spectrometer.

Mass spectrumMass spectrum

�� Proteins consist of 20 different types of a. a. with Proteins consist of 20 different types of a. a. with

different masses (except for one pair different masses (except for one pair LeuLeu and and IleIle))

�� Different peptides produce different spectraDifferent peptides produce different spectra

�� Use the spectrum of a peptide to determine its Use the spectrum of a peptide to determine its

sequencesequence

ObjectivesObjectives

�� Describe the steps of a typical peptide analysis Describe the steps of a typical peptide analysis

by MS (proteomic experiment)by MS (proteomic experiment)

�� Explain peptide ionization, fragmentation, Explain peptide ionization, fragmentation,

identificationidentification

Why are peptides, and not proteins, Why are peptides, and not proteins,

sequenced?sequenced?

�� Solubility under the same conditionsSolubility under the same conditions

�� Sensitivity of MS much higher for peptidesSensitivity of MS much higher for peptides

�� MS efficiencyMS efficiency

MS Peptide ExperimentMS Peptide Experiment

Choice of EnzymeChoice of Enzyme

XX--PhePhe, X, X--LeuLeu, X, X--IleIle, X, X--Met, XMet, X--Val, XVal, X--AlaAlaThermolysinThermolysin

PhePhe--X, X, TyrTyr--X, X, TrpTrp--X, X, LeuLeu--XXChymotrypsinChymotrypsin

B. NONSPECIFICB. NONSPECIFIC

XX--AspAspEndoproteinaseEndoproteinase AspAsp--NN

ArgArg--XXEndoproteinaseEndoproteinase ArgArg--CC

LysLys--XXEndoproteinaseEndoproteinase LysLys--CC

GluGlu--XXEndoproteinaseEndoproteinase GluGlu--CC

ArgArg--X, X, LysLys--XXTrypsinTrypsin

A. HIGHLY SPECIFICA. HIGHLY SPECIFIC

SpecificitySpecificityCleaving Cleaving

agent/Proteasesagent/Proteases

ESILiquid flow

Q or Ion Trapanalyzer

ESI is a solution technique that gives a continuous stream of ions,

best for quadrupoles, ion traps, etc.

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MALDI3 nS LASER PULSE

Sample (solid) on target at

high voltage/ high vacuum

MALDI is a solid-state technique that gives ions in pulses,

best suited to time-of-flight MS.

TOF analyzer

Atmosphere Low vac. High vac.

High vacuum

….MALDI or Electrospray ?

MALDI is limited to solid state, ESI to liquid

ESI is better for the analysis of complex mixture as it is

directly interfaced to a separation techniques (i.e. HPLC or

CE)

MALDI is more “flexible” (MW from 200 to 400,000 Da)

Q2Q2

Collision CellCollision CellQ3Q3

II

IIII

IIIIIICorrelative Correlative

sequence database sequence database

searchingsearching

TheoreticalTheoretical AcquiredAcquiredProtein identificationProtein identification

PeptidesPeptides

1D, 2D, 3D peptide separation1D, 2D, 3D peptide separation

200 400 600 80010001200m/zm/z

200 400 600 80010001200m/zm/z

200 400 600 80010001200m/zm/z

12 14 16

Time (min)

Tandem mass spectrumTandem mass spectrum

Protein Identification StrategyProtein Identification Strategy

Q1Q1

*

*

Protein Protein

mixturemixture

10-Mar-200514:28:10

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z0

100

%

CAL050310A 71 (1.353) Cm (1:96) TOF MSMS 785.60ES+ 2.94e3684.17

333.15

187.07

175.12

169.06

246.13

286.11

480.16

382.11

480.08

497.09

627.17

612.08

498.09

813.16

785.62

685.18

740.09

1285.141056.17942.16

814.17

924.16

943.17

1039.13

1038.17

1171.14

1057.18

1058.17

1172.15

1173.16

1286.14

1287.13

1296.10

10-Mar-200514:28:10

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z0

100

%

CAL050310A 71 (1.353) Cm (1:96) TOF MSMS 785.60ES+ 2.94e3684.17

333.15

187.07

175.12

169.06

246.13

286.11

480.16

382.11

480.08

497.09

627.17

612.08

498.09

813.16

785.62

685.18

740.09

1285.141056.17942.16

814.17

924.16

943.17

1039.13

1038.17

1171.14

1057.18

1058.17

1172.15

1173.16

1286.14

1287.13

1296.10

10-Mar-200514:28:10

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z0

100

%

CAL050310A 71 (1.353) Cm (1:96) TOF MSMS 785.60ES+ 2.94e3684.17

333.15

187.07

175.12

169.06

246.13

286.11

480.16

382.11

480.08

497.09

627.17

612.08

498.09

813.16

785.62

685.18

740.09

1285.141056.17942.16

814.17

924.16

943.17

1039.13

1038.17

1171.14

1057.18

1058.17

1172.15

1173.16

1286.14

1287.13

1296.10

Breaking Protein into Peptides and Breaking Protein into Peptides and

Peptides into Fragment IonsPeptides into Fragment Ions

�� Proteases, e.g. Proteases, e.g. trypsintrypsin, break protein into , break protein into

peptidespeptides

�� MS/MS breaks the peptides down into MS/MS breaks the peptides down into fragment fragment

ionsions and measures the mass of each pieceand measures the mass of each piece

�� MS measure MS measure m/zm/z ratio of an ionratio of an ion

Peptide fragmentationPeptide fragmentation

Amino acids

differ in their

side chains

Predominant

fragmentation

Weakest bonds

Tendency of peptides to fragment at Asp (D)

Mass Spectrometry in Proteomics

Ruedi Aebersold* and David R. Goodlett

269 Chem. Rev. 2001, 101, 269-295

C-terminal side of Asp

Large-scale Analysis of in Vivo Phosphorylated Membrane Proteins by Immobilized Metal Ion Affinity Chromatography and Mass

Spectrometry, Molecular & Cellular Proteomics, 2003, 2.11, 1234, Thomas S. Nuhse, Allan Stensballe, Ole N. Jensen, and Scott C.

Peck

What you need for peptide mass mappingWhat you need for peptide mass mapping

�� Peptide mass spectrumPeptide mass spectrum

�� Protein DatabaseProtein Database

�� GenBank, SwissGenBank, Swiss--Prot, dbEST, etc.Prot, dbEST, etc.

�� Search enginesSearch engines

�� MasCot, Prospector, Sequest, etc.MasCot, Prospector, Sequest, etc.

Database search for protein identification

Protein Identification by MS

Artificial spectra built

Artificially trypsinated

Database of sequences

(i.e. SwissProt)

Spot removed from gel

Fragmented using trypsin

Spectrum of fragments generated

MATCH

Library

ConclusionsConclusions

�� MS of peptides enables high throughput MS of peptides enables high throughput

identification and characterization of proteins in identification and characterization of proteins in

biological systemsbiological systems

�� ““de novo sequencingde novo sequencing”” can be used to identify can be used to identify

unknown proteins not found in protein databasesunknown proteins not found in protein databases

ReferencesReferencesH. Steen and M. Mann. H. Steen and M. Mann. ““The ABCThe ABC’’s (and s (and XYZXYZ’’ss) of Peptide ) of Peptide SequencingSequencing”” Molecular Cell Biology, Molecular Cell Biology, Nature ReviewsNature Reviews. . 2004, 5, 699.2004, 5, 699.

T. S. Nuhse, A. Stensballe, O. Jensen, and S. Peck. “Large-scale Analysis of in Vivo Phosphorylated Membrane Proteins by Immobilized Metal Ion Affinity Chromatography and Mass Spectrometry” Molecular & Cellular Proteomics, 2003, 2.11, 1234.

R. Aebersold and D. Goodlett. “Mass Spectrometry in Proteomics” Chem. Rev., 2001, 101, 269.