isobaric tags for quantitative analysis joshua j. coon u. wisconsin-madison
TRANSCRIPT
Isobaric tags for quantitative analysis
Joshua J. CoonU. Wisconsin-Madison
Isobaric Tagging
Ross,PL. et al MCP 2004
iTRAQ Structure
Available Reagents
Phosphorylation site on LIN28
Contemporary proteomics
Introduction of internal standard
Popular quantitative methods
MS1 vs MS2 quantitation
Number of quantitative data points
Dual cell linear ion trap orbitrap MS
Questions
Possible sources of error
1. Tag impurities
1. Tag impurities - Correction factors
1. Tag impurities - TagQuant
2. Low-intensity reporter ions
2. Low-intensity reporter ions – Summing
3. Interfering fragment ions
3. Sample Processing
4. Interference
4. Interference
Solutions to sources of error
Fragmentation method compatibility
ETD does not generate intense reporters
Gardner, Coon, Brodbelt, et al., Anal Chem, 2009
IRMPD on an dual-cell linear ion trap
IRMPD of isobaric tag-labeled peptide
Fragmentation method compatibility
Dual cell linear ion trap orbitrap MS
Performance improvements
McAlister, GC. et al Analytical Chemistry 2010
Metabolic labeling vs isobaric tags
Peptide Identification
Peptide and Protein Quantitation
Distribution of error
Triplicate Analyses
Triplicate Analyses
Quantitation validation
Samples
MS workflow
Results
Cell-type specific genes
Cell type specific genes
ES / iPS differences
Protein-protein interactions
Reprogramming networks
Triplicate 6-plex TMT experiment and correlation of protein to RNA
Ongoing 8-plex experiment
Software
Cyclin-dependent kinase 2
RNA up Protein down
1. Tag impurities
SRM and western blot validation