Why is Sequence Information Why is Sequence Information ImportantImportant

A protein’s amino acid sequence is A protein’s amino acid sequence is unique.unique. As little as 5 amino acid sequences As little as 5 amino acid sequences

can ID a proteincan ID a protein The sequence defines the primary The sequence defines the primary

structure of the proteinstructure of the protein the primary structure is the primary structure is

fundamental to understanding the fundamental to understanding the structure and function of the proteinstructure and function of the protein

The interrelationship between an The interrelationship between an amino acid sequence and the amino acid sequence and the corresponding DNA sequencecorresponding DNA sequence Protein sequences access gene Protein sequences access gene

sequences and are key to molecular sequences and are key to molecular biologybiology

Current Methods for Proteome Current Methods for Proteome ResearchResearch

SDS-PAGE SDS-PAGE separates based on molecular weight and/or separates based on molecular weight and/or

isoelectric pointisoelectric point 10 fmol - > 10 pmol sensitivity10 fmol - > 10 pmol sensitivity Tracks protein expression patternsTracks protein expression patterns

Protein SequencingProtein Sequencing Edman degradation or internal sequence analysisEdman degradation or internal sequence analysis

Immunological MethodsImmunological Methods Western BlotsWestern Blots

DrawbacksDrawbacks

SDS-Page can track the appearance, SDS-Page can track the appearance, disappearance or molecular weight shifts of disappearance or molecular weight shifts of proteins, but can not ID the protein or proteins, but can not ID the protein or measure the molecular weight with any measure the molecular weight with any accuracyaccuracy

Edman degradation requires a large amount Edman degradation requires a large amount of protein and does not work on N-terminal of protein and does not work on N-terminal blocked proteinsblocked proteins

Western blotting is presumptive, requires Western blotting is presumptive, requires the availability of suitable antibodies and the availability of suitable antibodies and have limited confidence in the ID related to have limited confidence in the ID related to the specificity of the antibody. the specificity of the antibody.

Advantageous of Mass Advantageous of Mass SpectrometrySpectrometry

Sensitivity in Sensitivity in attomole rangeattomole range

Rapid speed of Rapid speed of analysisanalysis

Ability to Ability to characterize and characterize and locate post-locate post-translational translational modificationsmodifications

Protein Identification Protein Identification ExperimentExperiment

Separated Proteins

Enzymatic Digestion and Extraction

MALDI-TOF Nano LC-MS-MS

Database Search Sequence Tag

Protein Identification

Database Search

Protein Identification

TrypsinTrypsin K-X and R-X except when X = K-X and R-X except when X = PP

Endoprotease Lys-CEndoprotease Lys-C K-X except when X = PK-X except when X = P

Endoprotease Arg-CEndoprotease Arg-C R-X except when X = PR-X except when X = P

Endoprotease Asp-NEndoprotease Asp-N X-DX-D

Endoprotease Glu-CEndoprotease Glu-C E-X except when X = PE-X except when X = P

ChymotrypsinChymotrypsin X-L, X-F, X-Y and X-WX-L, X-F, X-Y and X-W

Cyanogen BromideCyanogen Bromide X-MX-M

Enzymes for Proteome Research

Protease digestion

Protein Sample Peptides

m/z

MALDI Mass Spectrum

1000 2000

MeasureMeasured (Da)d (Da)

TheoreticTheoretical (Da)al (Da)

ErrorError ResiduesResidues SequenceSequence

1381.0101381.010 1380.7871380.787 0.2230.223 601-612601-612 QVLLHQQALFGKQVLLHQQALFGK

1400.884 1400.675 0.209 337-348 VVWCAVGPKKQK

1414.910 1414.752 0.158 322-333 NLRETAEEVKAR

1505.073 1505.073 0.249 302-315 IPSKVDSALYLGSR

1528.991 1528.991 0.221 337-349 VVWCAVGPEEQKK

1550.9851550.985 1550.9851550.985 0.2460.246 630-642630-642 NLLFNDNTECLAKNLLFNDNTECLAK

1725.1221725.122 1725.1221725.122 0.3010.301 667-681667-681 CSTSPLLEACAFLTRCSTSPLLEACAFLTR

1827.2121827.212 1827.2121827.212 0.3440.344 379-396379-396 GEADALNLDGGYIYTAGKGEADALNLDGGYIYTAGK

Peptides analyzed by MALDI

Micro-Sequencing by Tandem Micro-Sequencing by Tandem Mass Spectrometry (MS/MS)Mass Spectrometry (MS/MS)

Ions of interest are selected in the first mass Ions of interest are selected in the first mass analyzeranalyzer

Collision Induced Dissociation (CID) is used to Collision Induced Dissociation (CID) is used to fragment the selected ions by colliding the ions with fragment the selected ions by colliding the ions with gas (typically Argon for low energy CID)gas (typically Argon for low energy CID)

The second mass analyzer measures the fragment The second mass analyzer measures the fragment ionsions

The types of fragment ions observed in an MS/MS The types of fragment ions observed in an MS/MS spectrum depend on many factors including primary spectrum depend on many factors including primary sequence, the amount of internal energy, how the sequence, the amount of internal energy, how the energy was introduced, charge state, etc. energy was introduced, charge state, etc.

Fragmentation of peptides (amino acid chains) Fragmentation of peptides (amino acid chains) typically occurs along the peptide backbone. Each typically occurs along the peptide backbone. Each residue of the peptide chain successively fragments residue of the peptide chain successively fragments off, both in the N->C and C->N direction. off, both in the N->C and C->N direction.

Sequence Nomenclature Sequence Nomenclature for Mass Ladderfor Mass Ladder

H2NHC C

O

R1

HN

HC

R2

C

OHN

HC

R3

C

OHN CH

R4

C

O

OH

a1 a3a2 b3b2b1 c2c2c1

x1 x3x2 y3y2y1 z2z2z1

1598

14241295

1166

1052

965852

723

586

529401

Q G H E L S N E E R

+H

Roepstorff, P and Fohlman, J, Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed Mass Spectrom, 11(11) 601 (1984).

Protease digestion

Protein Sample Peptides

GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGFTYTDANKNKGITWKEETLMEYLENPKKYIPGTKMIFAGIKKKTEREDLIAYLKKATNE

m/z

First Stage Mass Spectrum

300 2200

TGPNLHGFGR

Selected Precursor mass and fragments

TGPNLHGLFGR

R

GR FGRGFGR etc

Protein Sequence

Peptides of precursors molecular weight fragmented

Peptides eluted from LC

Second Stage (fragmentation) Mass Spectrum

m/z75 2000

ReferencesReferences

Kinter, M.; Sherman, N. E. Kinter, M.; Sherman, N. E. Protein Protein Sequencing and Identification Using Sequencing and Identification Using Tandem Mass SpectrometryTandem Mass Spectrometry; Wiley-; Wiley-Interscience: New York, 2000.Interscience: New York, 2000.

Aebersold, R.; Mann, M. Aebersold, R.; Mann, M. NatureNature 20032003, , 422422, 198-207., 198-207.