PROTEIN IDENTIFICATION BY MASS SPECTROMETRY

OBJECTIVES To become familiar with matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) To become familiar with contemporary protein identification approaches To identify unknown proteins from collected samples by peptide mass fingerprinting, using mass spectral data of tryptic peptides for database querying

What is mass spectrometry ? Most accurate technique available to determine the mass of an analyte (proteins, peptides, small molecules)

Simple schematic of a mass spectrometer Mass AnalyzerIon Source Detector What is mass spectrometry ?

Basics of mass spectrometry Determination of mass to charge ratio (m/z) – note in MALDI-TOF MS, the charge is 1 so we essentially determine mass (i.e. m/1) Components of MS - ionization and entrance to gas phase - ion (peptide) separation - mass analysis

Output from MS - mass-to-charge ratio (m/z) ratio of exact mass to net charge For example: 802 m/z ion (z=1; +1 charged) = 802 amu 500 m/z ion (z=2; +2 charged) = 1000 amu 327 m/z ion (z=3; +3 charged) = ???? amu

Terminology Isotopes Ionization Resolution

ISOTOPES Atoms of an element with differing numbers of neutrons in their nuclei + n n n n n n n + n n n n n n 12 C 13 C 99% abundance1% abundance

10 carbons 10 x 12 C 9 x 12 C + 1 x 13 C 8 x 12 C + 2 x 13 C C1010/10/ :44:00 AM C10 Simulation C10 Profile Resolution: Daltons 0.25 at 5% height Charges 1 Chrg dist 0 Ions 11 Min Ion Ab. 1e-020 Min Ions 5000 Max Ions m/z Abundance

RESOLUTION How well two ions can be separated Good resolution essential to distinguishing isotopes Resolution usually decreases as the mass of the analyte increases

Res=2705 KISSME10/10/ :25:02 AM C28 H50 N7 O10 S1 Simulation KISSME+H Profile Resolution: Daltons 0.25 at 5% height Charges 1 Chrg dist 0 Ions Min Ion Ab. 1e-020 Min Ions 5000 Max Ions m/z Abundance

Res=901 KISSME10/10/ :25:45 AM C28 H50 N7 O10 S1 Simulation KISSME+H Profile Resolution: Daltons 0.75 at 5% height Charges 1 Chrg dist 0 Ions Min Ion Ab. 1e-020 Min Ions 5000 Max Ions m/z Abundance

Res=520 KISSME10/10/ :26:20 AM C28 H50 N7 O10 S1 Simulation KISSME+H Profile Resolution: Daltons 1.3 at 5% height Charges 1 Chrg dist 0 Ions Min Ion Ab. 1e-020 Min Ions 5000 Max Ions m/z Abundance

IONIZATION A + H + = (A+H) + A + Na + = (A+Na) + A - H = (A-H) - Giving an electric charge to a neutral species

Soft-ionization methods for proteomics MALDI –In MALDI-TOF MS, ionization occurs when the laser hits the sample Electrospray –In electrospray, ionization occurs in the liquid as it exits a spray needle

MALDI = Matrix-Assisted Laser Desorption / Ionization analyte is dried with matrix on target –matrix: CHCA, DHB, sinapinic acid –matrix absorbs laser energy and transfers to analyte generates predominantly singly-charged ions

MALDI = Matrix-Assisted Laser Desorption / Ionization A A A A A M M M M M M M M M M M M MM M M M M M M M M M M M M A A A A A A A A (ions and neutrals) laser A = analyte M = matrix

Mass Analyzers Time-Of-Flight (TOF) Quadrupole Ion Trap Ion Cyclotron Resonance

Mass to Charge Ratio (m/z) Measure m/z not mass –C7H7 mass is 91 Da –C7H7+1; m/z = 91 –C7H7+2; m/z = 45.5

Mass Analyzers: Reflectron Time-Of-Flight Ion Source Detector Electrostatic Mirror

MALDI-TOF mass spectrometers

MALDI-TOF MS matrix-assisted laser desorption / ionization- time of flight mass spectrometer Voyager DE-PRO Peptides put on to plate surface

Laser Sample plate Detector Mirror MALDI-TOF MS Design

Laser Sample plate Detector Mirror MALDI-TOF MS Design

Laser fires, clock starts Laser fires…Clock Starts Laser Analyte Sample plate Detector Mirror

Peptide ions are accelerated towards the mirror

Peptide ions enter the mirror

Peptide ions are reflected towards the detector

Larger ions take longer to “fly” from the sample plate to the detector

Peptide ions approach the detector

Mass scale Intensity scale Smallest peptide ion reaches the detector

mass intensity

mass intensity “Mass Spectrum” shows distribution and intensity of peptides in the sample

Mass (m/z) % intensity Mass Spectrum graph showing all peptides in the sample Small peptidesLarge peptides Reflector Spec #1 MC[BP = 842.5, 682]

Time of Flight Standard Mass Time of FlightMass? Mass (daltons) TOF (nanoseconds) How Time-Of-Flight works

Time of Flight Standard Mass Time of FlightMass? How Time-Of-Flight works Mass (daltons) TOF (nanoseconds)

Time of Flight Standard Mass Time of FlightMass? How Time-Of-Flight works Mass (daltons) TOF (nanoseconds)

Mass (daltons) TOF (nanoseconds) Time of Flight Standard Mass Time of FlightMass? How Time-Of-Flight works 5nsec = 1000 daltons

Time of Flight Standard Mass Time of FlightMass? How Time-Of-Flight works Mass (daltons) TOF (nanoseconds) 5nsec = 1000 daltons

Peptide mass fingerprinting (PMF) digestMS Search HIT SCORE Protein X 1000 Protein Y 50 Protein Z 5 Protein X theoretical digestProtein Y theoretical digestProtein Z theoretical digest Spectrum processing Report Protein (gel band or spot) peptides Mass spectrum Peak list

Computer program used to convert graph data (from mass spectrum) to table data (list of peptide masses) Generate mass list of all peptides

Mass (Da) Intensity Simple Mass Spectrum One protein, digested with trypsin, peptides extracted, analyzed by MALDI

PLEASEMAKEMANYKRISPYKREMETREATSTHEYAREGREAT Database search using computer algorithm List of masses e.g. MASCOT Sequence matches EAT EGR ISPYK EMETR EMANYK PLEASEMAK EATSTHEYAR

QUESTIONS?