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Techniques for the analysis of proteins and proteomes Rainer Breitling Groningen Bioinformatics Centre
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Lecture-9MS Techniques and Protein
Identification
Huseyin Tombuloglu, Phd
GBE423 Genomics & Proteomics
• Diversity of Proteins
• Separating Proteins
• 2D gel electrophoresis, mass spectrometry
• Identifying Proteins
• peptide fingerprinting, de novo sequencing
• Quantifying Proteins
• DIGE, ICAT
• Protein interactions
• high-throughput mass spectrometry
Techniques for the analysis of proteins and proteomes
Rainer BreitlingGroningen Bioinformatics Centre
Diversity of proteins
Hydrophobicity
Polarity
Acidity
Charge
Size
Proteins are much more diverse
Diversity of proteins
• More than 5% of all genes can produce variant proteins by alternative splicing.
Diversity of proteinsProtein targeting and trafficking:
• to nucleus or mitochondria
• to vesicular apparatus
• to peroxisomes
• to membranes and extracellular space
Diversity of proteins• phosphorylation, the addition of a phosphate group, usually to
serine, tyrosine, threonine or histidine kinase signalling cascades• acetylation, the addition of an acetyl group, usually at the N-
terminus of the protein • alkylation, the addition of an alkyl group (e.g. methyl, ethyl) • methylation the addition of a methyl group, usually at lysine or
arginine residues (this is a subtype of alkylation) histone modification
• isoprenylation, the addition of an isoprenoid group (e.g. farnesol and geranylgeraniol) membrane targeting
• glycosylation, the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein extracellular stability
• Tyrosine sulfation, the addition of a sulfate group to a tyrosine. • ubiquitination, the covalent linkage of the protein ubiquitin to a target
protein protein degradation/trafficking• SUMOylation, the covalent linkage of the SUMO protein (Small
Ubiquitin-related MOdifier) to a target protein.
Conventional Proteomic Approach
Excise gel spot
Tryptic digest
2D-PAGE
Destain
Proteins from lysed cell
Reduce w/DTT
Alkylate w/IAA
OR
Matrix Assisted Laser Desorption Ionization -Mass Spectrometry
ElectrosprayIonization –Mass Spectrometry
2D gel electrophoresis
isoelectric focusing separation by pI
SD
S g
el e
lect
roph
ores
is
sep
arat
ion
by m
ass
2D gel electrophoresis
Control Experimental
2D gel electrophoresis
Control
Experimental
PTM?
Downregulation?
Mass Spectrometer
Ionizer
Sample
+_
Mass Analyzer Detector• MALDI• Electro-Spray
Ionization (ESI)
• Time-Of-Flight (TOF)• Quadrupole• Ion-Trap
• ElectronMultiplier(EM)
MALDI-TOF-MS
Mass Spectrometer (MALDI-TOF)
Source
Length = s
Field-free drift zone
Length = D
Ed = 0
Microchannel plate detector
Backing plate(grounded) Extraction grid
(source voltage -Vs)
UV (337 nm)
Detector grid -Vs
Pulse voltage
Analyte/matrix
Electrospray Ionization-MS
Quadrupole time-of-flight (Q-TOF)
CID spectra (collision induced dissociation) are obtained from the MS/MS mass analyzer.
Ion Source
Mass Analyzer
Detector
(2) Aebersold, R.; Mann, M. Nature 2003, 422, 198-207.
Peptide Mass Fingerprint
Trypsin Digest
This section is taken from Nathan Edwards, UMIACS
Peptide Mass Fingerprint
MS
Peptide Mass Fingerprint• Trypsin: digestion enzyme
– Highly specific– Cuts after K & R except if followed by P
• Protein sequence from sequence database– In silico digest– Mass computation
• For each protein sequence in turn:– Compare computer generated masses with observed
spectrum
Protein Sequence
• Myoglobin - Plains zebra
GLSDGEWQQV LNVWGKVEAD IAGHGQEVLI RLFTGHPETL EKFDKFKHLK TEAEMKASED LKKHGTVVLT ALGGILKKKG HHEAELKPLA QSHATKHKIP IKYLEFISDA IIHVLHSKHP GDFGADAQGA MTKALELFRN DIAAKYKELG FQG
Protein Sequence
• Myoglobin - Plains zebra
GLSDGEWQQV LNVWGKVEAD IAGHGQEVLI RLFTGHPETL EKFDKFKHLK TEAEMKASED LKKHGTVVLT ALGGILKKKG HHEAELKPLA QSHATKHKIP IKYLEFISDA IIHVLHSKHP GDFGADAQGA MTKALELFRN DIAAKYKELG FQG
Peptide Masses
1811.90 GLSDGEWQQVLNVWGK 1606.85 VEADIAGHGQEVLIR 1271.66 LFTGHPETLEK 1378.83 HGTVVLTALGGILK 1982.05 KGHHEAELKPLAQSHATK 1853.95 GHHEAELKPLAQSHATK 1884.01 YLEFISDAIIHVLHSK 1502.66 HPGDFGADAQGAMTK 748.43 ALELFR
Peptide Mass Fingerprint
GLS
DG
EWQ
QVL
NVW
GK
VEA
DIA
GH
GQ
EVLI
R
LFTG
HPE
TLEK
HG
TVVL
TALG
GIL
K
KG
HH
EAEL
KPL
AQ
SHA
TK
GH
HEA
ELK
PLA
QSH
ATK
YLEF
ISD
AIIH
VLH
SK
HPG
DFG
AD
AQ
GA
MTK
ALE
LFR
Mass Spectrometry
• Strengths– Precise molecular weight– Fragmentation– Automated
• Weaknesses– Best for a few molecules at a time– Best for small molecules– Mass-to-charge ratio, not mass– Intensity ≠ Abundance
Single Stage MS
MS
Tandem Mass Spectrometry(MS/MS)
Precursor selection
Tandem Mass Spectrometry(MS/MS)
Precursor selection + collision induced dissociation
(CID)
MS/MS
Peptide Fragmentation
H…-HN-CH-CO-NH-CH-CO-NH-CH-CO-…OH
Ri-1 Ri Ri+1
AA residuei-1 AA residuei AA residuei+1
N-terminus
C-terminus
Peptides consist of amino-acids arranged in a linear backbone.
i+1
Peptide Fragmentation
-HN-CH-CO-NH-CH-CO-NH-
Ri CH-R’
bi
yn-iyn-i-1
bi+1
R”i+1
Peptide Fragmentation
Peptide: S-G-F-L-E-E-D-E-L-KMW ion ion MW
88 b1 S GFLEEDELK y9 1080
145 b2 SG FLEEDELK y8 1022
292 b3 SGF LEEDELK y7 875
405 b4 SGFL EEDELK y6 762
534 b5 SGFLE EDELK y5 633
663 b6 SGFLEE DELK y4 504
778 b7 SGFLEED ELK y3 389
907 b8 SGFLEEDE LK y2 260
1020 b9 SGFLEEDEL K y1 147
Peptide Fragmentation
100
0250 500 750 1000 m/z
% In
tens
ity
K1166
L1020
E907
D778
E663
E534
L405
F292
G145
S88 b ions
147260389504633762875102210801166 y ions
Peptide Fragmentation
K1166
L1020
E907
D778
E663
E534
L405
F292
G145
S88 b ions
100
0250 500 750 1000 m/z
% In
tens
ity
147260389504633762875102210801166 y ionsy6
y7
y2 y3 y4
y5
y8 y9
b3
b5 b6 b7b8 b9
b4
Peptide Identification
Given:• The mass of the precursor ion, and• The MS/MS spectrum
Output:• The amino-acid sequence of the peptide
Sequence Database Search
• Sequence fills in gaps in the spectrum• All candidates have biological relevance• Practical for high-throughput peptide
identification• Correct peptide might be missing from
database!
Mascot Search Engine
Mascot MS/MS Ions Search
Mascot MS/MS Search Results
Mascot MS/MS Search Results
Mascot MS/MS Search Results
