Isobaric tags for quantitative analysis Joshua J. Coon U. Wisconsin-Madison

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