Evaluation of the Potential Risk of Advanced Peak Determination in Distorting Isobaric Labeling-Based Single-Shot Proteome Quantitation.

: The recent development and implementation of the advanced peak determination (APD) algorithm with MS instrument dramatically increased the sampling of low abundance features for MS/MS fragmentation. After in-depth evaluation, we found that with APD on many chimeric spectra were acquired through co-fragmentation of high abundance contaminants with low abundance targets, and such co-fragmentations were largely avoided when APD was off. To evaluate whether such a co-fragmentation could significantly distort the accuracy of the isobaric-labeling based quantitation of the low abundance target, we performed a single-shot TMT experiment using a two-proteome model, whereby each TMT channel contains premixed peptides from human and a cyanobacterium with a known ratio. Unexpectedly, we found that APD did not significantly distort TMT ratios, probably because the majority of the APD-specific chimeric spectra were not identifiable. Nevertheless, we found a few examples of significant distortion of TMT ratios of low abundance peptides caused by APD through manual inspection, and suggested that APD should be off in a single-shot TMT experiment to avoid the laborious and time-costing manual inspection. This article is protected by copyright. All rights reserved.