Peptide Sequencing by Peptide Sequencing by Mass SpectrometryMass Spectrometry

Alex RamosAlex Ramos5 April 20055 April 2005

Edman degradationEdman degradation

N C S H2N C

H

CH3

C

O

Asp Phe Phe Arg CO

O-+

N C

H

CH3

C

O

Asp Phe Phe Arg CO

O-C

S H

N

H

Labeling

NN

S

O

CH3

H

PTH-alanine

Asp Phe Phe Arg CO

O-H2N+

Release

Peptide shorthened by one residue

Phenyl isothiocyanate

Edman Degradation v. MS/MSEdman Degradation 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 Edman DegradationEdman Degradation 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 (“de novo Derivation without help from a protein database (“de novo sequencing”), especially important in identification of sequencing”), 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 peptides2. Peptides charged (ionized) and separated according 2. Peptides charged (ionized) and separated according

to their different m/z ratiosto their different m/z ratios3. Each peptide fragmented into ions and m/z values of 3. Each peptide fragmented into ions and m/z 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 Leu and different masses (except for one pair Leu and Ile)Ile)

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

Cleaving Cleaving agent/Proteasesagent/Proteases

SpecificitySpecificity

A. HIGHLY SPECIFICA. HIGHLY SPECIFIC

TrypsinTrypsin Arg-X, Lys-XArg-X, Lys-X

Endoproteinase Glu-CEndoproteinase Glu-C Glu-XGlu-X

Endoproteinase Lys-CEndoproteinase Lys-C Lys-XLys-X

Endoproteinase Arg-CEndoproteinase Arg-C Arg-XArg-X

Endoproteinase Asp-NEndoproteinase Asp-N X-AspX-Asp

B. NONSPECIFICB. NONSPECIFIC

ChymotrypsinChymotrypsin Phe-X, Tyr-X, Trp-X, Leu-XPhe-X, Tyr-X, Trp-X, Leu-X

ThermolysinThermolysin X-Phe, X-Leu, X-Ile, X-Met, X-Val, X-AlaX-Phe, X-Leu, X-Ile, X-Met, X-Val, X-Ala

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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++

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++

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++

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+++

++

+

+++

++

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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)

Q2Q2Collision CellCollision Cell

Q3Q3

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 16Time (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

627.17612.08

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

627.17612.08

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

627.17612.08

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. trypsin, break protein into Proteases, e.g. trypsin, 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 piece and measures the mass of each piece

MS measure m/z ratio of an ionMS measure m/z ratio of an ion

Peptide fragmentationPeptide fragmentationAmino acids differ in their side chains

Predominant fragmentation

Weakest bonds

Tendency of peptides to fragment at Asp (D)

Mass Spectrometry in ProteomicsRuedi Aebersold* and David R. Goodlett269 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, Swiss-Prot, dbEST, etc.GenBank, Swiss-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

MATCHLi

bra

ry

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 sequencing” can be used to identify de novo sequencing” can be used to identify unknown proteins not found in protein databasesunknown proteins not found in protein databases

ReferencesReferencesH. Steen and M. Mann. “The ABC’s (and XYZ’s) of Peptide H. Steen and M. Mann. “The ABC’s (and XYZ’s) of Peptide Sequencing” Molecular Cell Biology, Sequencing” 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.