Technical, experimental, and biological variations in isobaric tags for relative and absolute quantitation (iTRAQ).
We assess the reliability of isobaric-tags for relative and absolute quantitation (iTRAQ), based on different types of replicate analyses taking into account technical, experimental, and biological variations. In total, 10 iTRAQ experiments were analyzed across three domains of life involving Saccharomyces cerevisiae KAY446, Sulfolobus solfataricus P2, and Synechocystis sp. PCC 6803. The coverage of protein expression of iTRAQ analysis increases as the variation tolerance increases. In brief, a cutoff point at +/-50% variation (+/-0.50) would yield 88% coverage in quantification based on an analysis of biological replicates. Technical replicate analysis produces a higher coverage level of 95% at a lower cutoff point of +/-30% variation. Experimental or iTRAQ variations exhibit similar behavior as biological variations, which suggest that most of the measurable deviations come from biological variations. These findings underline the importance of replicate analysis as a validation tool and benchmarking technique in protein expression analysis.
Gan
Chee Sian
CS
Biological and Environmental Systems Group, Department of Chemical and Process Engineering, The University of Sheffield, Mappin Street, Sheffield S1 3JD, United Kingdom.
Chong
Poh Kuan
PK
Pham
Trong Khoa
TK
Wright
Phillip C
PC
D019843:Archaeal Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007202:Indicators and Reagents; D013058:Mass Spectrometry / Q000379:methods; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000254:growth & development*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D048229:Sulfolobus solfataricus / Q000254:growth & development*; D046939:Synechocystis / Q000254:growth & development*
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D007202:Indicators and Reagents; D029701:Saccharomyces cerevisiae Proteins