iTRAQ experimental design for plasma biomarker discovery.
There is considerable interest in using mass spectrometry for biomarker discovery in human blood plasma. We investigated aspects of experimental design for large studies that require analysis of multiple sample sets using iTRAQ reagents for sample multiplexing and quantitation. Immunodepleted plasma samples from healthy volunteers were compared to immunodepleted plasma from patients with osteoarthritis in eight separate iTRAQ experiments. Our analyses utilizing ProteinPilot software for peptide identification and quantitation showed that the methodology afforded excellent reproducibility from run to run for determining protein level ratios (coefficient of variation 11.7%), in spite of considerable quantitative variances observed between different peptides for a given protein. Peptides with high variances were associated with lower intensity iTRAQ reporter ions, while immunodepletion prior to sample analysis had a negligible affect on quantitative variance. We examined the influence of different reference samples, such as pooled samples or individual samples on calculating quantitative ratios. Our findings are discussed in the context of optimizing iTRAQ experimental design for robust plasma-based biomarker discovery.
Australian Proteome Analysis Facility Ltd., Macquarie University, Sydney, Australia.
Xiaomin
X
Song
Julia
J
Bandow
Jamie
J
Sherman
J David
JD
Baker
Paul W
PW
Brown
Michael T
MT
McDowell
Mark P
MP
Molloy
D015415:Biomarkers / Q000097:blood*; D002411:Cation Exchange Resins; D002852:Chromatography, Ion Exchange; D005260:Female; D006801:Humans; D010003:Osteoarthritis / Q000378:metabolism; D010949:Plasma; D011933:Reagent Kits, Diagnostic / Q000592:standards; D012015:Reference Standards; D012016:Reference Values; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D015415:Biomarkers; D002411:Cation Exchange Resins; D011933:Reagent Kits, Diagnostic