lecture-9 ms techniques and protein identification huseyin tombuloglu, phd gbe423 genomics &...

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