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