| Abstract: | 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, it is found that with APD on, many chimeric spectra are acquired through co‐fragmentation of high abundance contaminants with low abundance targets, and such co‐fragmentations are largely avoided when APD is off. To evaluate whether such a co‐fragmentation could significantly distort the accuracy of the isobaric‐labeling based quantitation of the low abundance target, a single‐shot TMT experiment is performed using a two‐proteome model, whereby each TMT channel contains premixed peptides from human and a cyanobacterium with a known ratio. Unexpectedly, it is found that APD does not significantly distort TMT ratios, probably because the majority of the APD‐specific chimeric spectra are not identifiable. Nevertheless, a few examples of significant distortion of TMT ratios of low abundance peptides caused by APD is found through manual inspection, and suggests that APD should be off in a single‐shot TMT experiment to avoid the laborious and time‐costing manual inspection. |
