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Published byHolly Newman Modified over 9 years ago
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Isobaric tags for quantitative analysis Joshua J. Coon U. Wisconsin-Madison
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Isobaric Tagging Ross,PL. et al MCP 2004
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iTRAQ Structure
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Available Reagents
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Phosphorylation site on LIN28
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Contemporary proteomics
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Introduction of internal standard
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Popular quantitative methods
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MS1 vs MS2 quantitation
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Number of quantitative data points
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Dual cell linear ion trap orbitrap MS
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Questions
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Possible sources of error
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1. Tag impurities
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1. Tag impurities - Correction factors
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1. Tag impurities - TagQuant
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2. Low-intensity reporter ions
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2. Low-intensity reporter ions – Summing
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3. Interfering fragment ions
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3. Sample Processing
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4. Interference
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Solutions to sources of error
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Fragmentation method compatibility
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ETD does not generate intense reporters
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Gardner, Coon, Brodbelt, et al., Anal Chem, 2009 IRMPD on an dual-cell linear ion trap
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IRMPD of isobaric tag-labeled peptide
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Fragmentation method compatibility
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Dual cell linear ion trap orbitrap MS
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Performance improvements McAlister, GC. et al Analytical Chemistry 2010
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Metabolic labeling vs isobaric tags
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Peptide Identification
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Peptide and Protein Quantitation
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Distribution of error
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Triplicate Analyses
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Quantitation validation
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Samples
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MS workflow
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Results
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Cell-type specific genes
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Cell type specific genes
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ES / iPS differences
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Protein-protein interactions
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Reprogramming networks
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Triplicate 6-plex TMT experiment and correlation of protein to RNA
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Ongoing 8-plex experiment
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Software
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Cyclin-dependent kinase 2
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RNA up Protein down
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1. Tag impurities
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SRM and western blot validation
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