peptide sequencing by mass spectrometry
DESCRIPTION
Peptide Sequencing by Mass Spectrometry. Alex Ramos 5 April 2005. Edman degradation. Edman Degradation v. MS/MS. Why study proteins?. machines that make cells function RNA levels do not always accurately predict protein levels targets of drugs. Edman Degradation MS More sensitive - PowerPoint PPT PresentationTRANSCRIPT
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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+++ ++++++ ++++++ ++++++ ++++++ ++++++ ++++++ ++++++ +++ +
+++++++++++++++++++++++
++++++++++++++++++++++++
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.