Post on 15-Mar-2018
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
H
C H 3
C
O
A sp P he P he A rg C
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O-
+
N C
H
C H 3
C
O
A sp P he P he A rg C
O
O-
C
S H
N
H
L a be ling
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N
S
O
C H 3
H
PT H -a la nine
A sp P he P he A rg C
O
O-
H 2N+
R e le a s e
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.