Analysis of Reproducibility of Proteome Coverage and Quantitation Using Isobaric Mass Tags (iTRAQ and TMT)

This
study aimed to compare the depth and reproducibility of total
proteome and differentially expressed protein coverage in technical
duplicates and triplicates using iTRAQ 4-plex, iTRAQ 8-plex, and TMT
6-plex reagents. The analysis was undertaken because comprehensive
comparisons of isobaric mass tag reproducibility have not been widely
reported in the literature. The highest number of proteins was identified
with 4-plex, followed by 8-plex and then 6-plex reagents. Quantitative
analyses revealed that more differentially expressed proteins were
identified with 4-plex reagents than 8-plex reagents and 6-plex reagents.
Replicate reproducibility was determined to be ≥69% for technical
duplicates and ≥57% for technical triplicates. The results
indicate that running an 8-plex or 6-plex experiment instead of a
4-plex experiment resulted in 26 or 39% fewer protein identifications,
respectively. When 4-plex spectra were searched with three software
toolsProteinPilot, Mascot, and Proteome Discovererthe
highest number of protein identifications were obtained with Mascot.
The analysis of negative controls demonstrated the importance of running
experiments as replicates. Overall, this study demonstrates the advantages
of using iTRAQ 4-plex reagents over iTRAQ 8-plex and TMT 6-plex reagents,
provides estimates of technical duplicate and triplicate reproducibility,
and emphasizes the value of running replicate samples.