Synthetic Knockout Protein Standard for Evaluating Interference in Tandem Mass Tag-Based Proteomics

Isobaric labeling is widely used for unbiased, proteome-wide
studies,
and it provides several advantages, such as fewer missing values among
samples and higher quantitative precision. However, ion interference
may lead to compressed or distorted observed ratios due to the coelution
and coanalysis of peptides. Here, we introduced a synthetic KnockOut
standard (sKO) for evaluating interference in tandem mass tags-based
proteomics. sKO is made by mixing TMTpro-labeled tryptic peptides
derived from four nonhuman proteins and a whole human proteome as
background at different proportions. We showcased the utility of the
sKO standard by exploring ion interference at different peptide concentrations
(up to a 30-fold change in abundance) and using a variety of mass
spectrometer data acquisition strategies. We also demonstrated that
the sKO standard could provide valuable information for the rational
design of acquisition strategies to achieve optimal data quality and
discussed its potential applications for high-throughput proteomics
workflows development.