Balancing robust quantification and identification for iTRAQ: application of UHR-ToF MS.

ABSTRACT

iTRAQ reagents allow the simultaneous multiplex identification and quantification of a large number of proteins. Success depends on effective peptide fragmentation in order to generate both peptide sequence ions (higher mass region, 150-2200 m/z) and reporter ions (low mass region, 113-121 m/z) for protein identification and relative quantification, respectively. After collision-induced dissociation, the key requirements to achieve a good balance between the high and low m/z ions are effective ion transmission and detection across the MS/MS mass range, since the ion transmission of the higher m/z range competes with that of the low m/z range. This study describes an analytical strategy for the implementation of iTRAQ on maXis UHR-Qq-ToF instruments, and discusses the impact of adjusting the MS/MS ion transmission parameters on the quality of the overall data sets. A technical discussion highlights a number of maXis-specific parameters, their impact of quantification and identification, and their cross-interactions.