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 )