absolute quantification of proteins and phosphoproteins from cell lysates by tandem ms gygi et al...
TRANSCRIPT
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 amino acid sequence protease
solid phase peptide synthesis
incorporate stable isotopes
evaluate peptide by LC-MS/MS
AQUA = absolute quantification
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 LC-MS/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 known proteins Y-type fragment ions for SRM
Improvements high throughput
For unknown proteins