Vermont Genetics Network OutreachVermont Genetics Network Outreach
Proteomics Module Proteomics Module
Protein Mass Spectrometry:Protein Mass Spectrometry:
Theory and ApplicationTheory and Application
Prepared by Bryan A. Ballif, Ph.D.Prepared by Bryan A. Ballif, Ph.D.
Two Essential Partner Tools in ProteomicsTwo Essential Partner Tools in Proteomics
Mass SpectrometryMass SpectrometryGel ElectrophoresisGel Electrophoresis
First Things First -- Know Your Goal !First Things First -- Know Your Goal !
● ● There are nearly as many mass spectrometry There are nearly as many mass spectrometry methods as there are mass spectrometry projects. methods as there are mass spectrometry projects.
● ● Your goal determines your methods which determineYour goal determines your methods which determineyour outcomes.your outcomes.
The Four Most Common Protein Mass Spectrometry ProjectsThe Four Most Common Protein Mass Spectrometry Projects(Observe How Each Project Has A Distinct Goal / Method !!)(Observe How Each Project Has A Distinct Goal / Method !!)
What is this What is this protein?protein?
What proteins are What proteins are present in _____?present in _____?
RateCSF
Where Is this proteinWhere Is this proteinPost-translationallyPost-translationally
Modified?Modified?
Stimulus
Block
WesternBlotα-pS
What proteinWhat proteinChanges occur Changes occur
Following ______?Following ______?
StimulusH2O2
Block
WesternBlotα-pY
Purify pY ProteinsIdentify pY ProteinsIdentify pY SitesQuantify pY Changes
Typical Protein Mass Spectrometry Work FlowTypical Protein Mass Spectrometry Work Flow
Protein Preparation / Protein Preparation / Analyte PreparationAnalyte Preparation
Data Analysis /Data Analysis /InterpretationInterpretation
Mass SpectrometryMass Spectrometry
Junk In …Junk In …
Junk Out !Junk Out !
HPLCHPLCpumpspumps
Most Proteomics Experiments FurtherMost Proteomics Experiments FurtherPurify the Analyte Using Purify the Analyte Using
High Performance Liquid Chromatography (HPLC)High Performance Liquid Chromatography (HPLC)
H2OH2OOrganicOrganicSolventSolvent
Mass SpectrometerMass Spectrometer
Carbon ColumnCarbon Column
Electric / Magnetic Electric / Magnetic FieldsFields
Two Common Types of Peptide IonizationTwo Common Types of Peptide Ionization
Electrospray IonizationElectrospray IonizationMatrix-Assisted Laser Matrix-Assisted Laser
Desorption Ionization (MALDI)Desorption Ionization (MALDI)
John Fenn John Fenn (2002 Nobel Prize)(2002 Nobel Prize)
Koichi TanakaKoichi Tanaka(2002 Nobel Prize)(2002 Nobel Prize)
Image from Kore Technology Limited
Time of Flight Mass SpectrometryTime of Flight Mass Spectrometry
Mass Spectrometer
Carbon Column
Electric / Magnetic Fields
Perhaps the Most Simple Concept forPerhaps the Most Simple Concept forMass Measurements in Mass SpectrometerMass Measurements in Mass Spectrometer
Remember there are relationships between mass and Energy : Remember there are relationships between mass and Energy : E = mcE = mc2 2 or E or Ekk = ½ mv = ½ mv22
Electric field gives ions defined kinetic energy.
EEkinetickinetic = ½ mv = ½ mv22
Peptides (Bottom Up Proteomics) Peptides (Bottom Up Proteomics) Versus Protein (Top Down)Versus Protein (Top Down)
Tryptic DigestTryptic Digest
MFSCFLQAGNPQGSMFSCFLQAGNPQGSRRSGFGHNVELVSGFGHNVELVRRHASIWVTYHSEEHASIWVTYHSEEKKLLIPYSDELLLIPYSDEL
MFSCFLQAGNPQGSMFSCFLQAGNPQGSRR
SGFGHNVELVSGFGHNVELVRR
HASIWVTYHSEEHASIWVTYHSEEKK
LLIPYSDELLLIPYSDEL
NH3 G-F-S-F-P-V-A-T-G-L-M-E-D COOHD-G-K-P-R-
Another Problem: Another Problem: Multiple Peptides may have the same Mass! Multiple Peptides may have the same Mass!
NH3 G-F-S-F-P-M-L-G-T-A-V-E-D COOHD-G-K-P-R-
HPLCHPLCpumpspumps
MSMS11 MSMS22
PeptidePeptideIonsIons
PeptidePeptideFragmentFragmentIonsIons
Basics of Liquid Chromatography (LC) Basics of Liquid Chromatography (LC) Tandem Mass Spectrometry (MS/MS) for PeptidesTandem Mass Spectrometry (MS/MS) for Peptides
Mass SpectrometerMass Spectrometer
Rel
ativ
e A
bu
nd
ance
Rel
ativ
e A
bu
nd
ance
timetime
organic organic concentration in concentration in mobile phasemobile phase
Rel
ativ
e A
bu
nd
ance
Rel
ativ
e A
bu
nd
ance
m/zm/z
LCLC
m/zm/zlowlow highhigh
Knowing your ABC’s and your XYZ’s:Knowing your ABC’s and your XYZ’s:In your “Daughter” or “Fragment” IonsIn your “Daughter” or “Fragment” Ions
-12345 Y ions
1 2 3 4 5 6 7 8 9 10 11 12 13b ionsG-F-S-F-P-V-A-T-G-L-M-E-D-D-G-K-P-R COOHNH3
He
NH3 G-F-S-F-P-V-A-T-G-L-M-E-D COOHD-G-K-P-R
A Closer Look at MSA Closer Look at MS2 2 (For Peptide Identification) (For Peptide Identification)
12345 Y ions678
1 2 3 4 5 6 7 8 9 10b ions
He
Collision-Collision-InducedInduced
DissociationDissociation( CID )( CID )
G-F-S-F-P-V-A-T-G-L-M-E-D-D-G-K-P-RCOOHNH3
P
He
-
A Closer Look at MSA Closer Look at MS22 (and Phosphorylation ) (and Phosphorylation )
