Absolute quantification of proteins and phosphoproteins from cell

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Transcript Absolute quantification of proteins and phosphoproteins from cell

Absolute quantification of
proteins and phosphoproteins
from cell lysates by tandem MS
Gygi et al (2003) PNAS
100(12), 6940-6945.
presented by Jessica Lee for
MEDG 505
Outline
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Introduction
Methods & Results
Conclusions
Critiques
Discussion Topics
Previous methods
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Relative quantification
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2-dimensional gel electrophoresis
tandem MS
Absolute quantification
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use of synthetic peptides with the stable
isotopes incorporated.
Previous methods
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Two-dimensional gel electrophoresis
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quantification by protein staining
comparative analysis
protein identified by MS
limited to abundant proteins
Previous methods
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Tandem MS
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protein and peptide separation coupled
with amino acid sequence analysis
quantification by stable isotopes labelling
mass accuracy
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resolution
reliability of the mass calibration relationship
Previous methods
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Absolute protein quantification with
synthetic peptide
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stable isotopes incorporation
liquid chromatography (LC) – MS
selected reaction monitoring (SRM)
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extreme sensitivity
target -> precursor ion -> production ion
Methods
Part 1: AQUA internal standard
peptide
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chose peptide based on
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AQUA = absolute quantification
amino acid sequence
protease
solid phase peptide
synthesis
incorporate stable
isotopes
evaluate peptide by LCMS/MS
Part 2: method development &
implemmentation
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separate whole cell
lysate by SDS/PAGE gel
electrophoresis
in gel digestion with the
AQUA peptides
LC-SRM
determine precise
expression level from LCMS/MS
Validations
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Purified protein
Target protein in whole cell lysate
Trypsinization
Low-abundance proteins in whole cell
lysate
Posttranslational modified protein phosphorylation
Conclusions
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Sensitivity and specificity
Sample requirement
Cover the short comings of previous
methods
Assist the study of systems biology in
diverse ways
Insights into cellular events and
pathways
Critiques
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Lack of information
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introduction
discussion
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limitation
Short comings of previous methods
Complement to lack of protein
abundance measurement
Discussion topics
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Limitations of this methods
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Improvements
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known proteins
Y-type fragment ions for SRM
high throughput
For unknown proteins