Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Reproducibility: Implication of Multiple Injections

We analyzed 10 isobaric tags for relative and absolute quantitation (iTRAQ) experiments using three
different model organisms across the domains of life:  Saccharomyces cerevisiae KAY446, Sulfolobus
solfataricus P2, and Synechocystis sp. PCC6803. A double database search strategy was employed to
minimize the rate of false positives to less than 3% for all organisms. The reliability of proteins with
single-peptide identification was also assessed using the search strategy, coupled with multiple analyses
of samples into LC−MS/MS. The outcomes of the three LC−MS/MS analyses provided higher proteome
coverage with an average increment in total proteins identified of 6%, 33%, and 50% found in S.
cerevisiae, S. solfataricus, and Synechocystis sp., respectively. The iTRAQ quantification values were
found to be highly reproducible across the injections, with an average coefficient of variation (CV) of
0.09 (scattering from 0.14 to 0.04) calculated based on log mean average ratio for all three organisms.
Hence, we recommend multiple analyses of iTRAQ samples for greater proteome coverage and precise
quantification.
Keywords: iTRAQ • mass spectrometer • multiple injections • double database search strategy • protein quantification
• reproducibility