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
Introduction
Methods & Results
Conclusions
Critiques
Discussion Topics
Previous methods
Relative quantification
2-dimensional gel electrophoresis
tandem MS
Absolute quantification
use of synthetic peptides with the stable
isotopes incorporated.
Previous methods
Two-dimensional gel electrophoresis
quantification by protein staining
comparative analysis
protein identified by MS
limited to abundant proteins
Previous methods
Tandem MS
protein and peptide separation coupled
with amino acid sequence analysis
quantification by stable isotopes labelling
mass accuracy
resolution
reliability of the mass calibration relationship
Previous methods
Absolute protein quantification with
synthetic peptide
stable isotopes incorporation
liquid chromatography (LC) – MS
selected reaction monitoring (SRM)
extreme sensitivity
target -> precursor ion -> production ion
Methods
Part 1: AQUA internal standard
peptide
chose peptide based on
AQUA = absolute quantification
amino acid sequence
protease
solid phase peptide
synthesis
incorporate stable
isotopes
evaluate peptide by LCMS/MS
Part 2: method development &
implemmentation
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
Purified protein
Target protein in whole cell lysate
Trypsinization
Low-abundance proteins in whole cell
lysate
Posttranslational modified protein phosphorylation
Conclusions
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
Lack of information
introduction
discussion
limitation
Short comings of previous methods
Complement to lack of protein
abundance measurement
Discussion topics
Limitations of this methods
Improvements
known proteins
Y-type fragment ions for SRM
high throughput
For unknown proteins