HYpro16: A Two-Proteome Mixture to Assess Interference in Isobaric Tag-Based Sample Multiplexing Experiments.

Isobaric tagging is a powerful strategy for global proteome profiling. A caveat of isobaric-tag-based quantification is "interference", which may be caused by coeluting peptides that are coisolated, cofragmented, and coanalyzed, thereby confounding quantitative accuracy. Here, we present a two-proteome standard that challenges the mass spectrometer to measure a range of protein abundance ratios in a background of potential interference. The HYpro16 standard consists of tandem mass tag (TMT) pro16-labeled human peptides at a 1:1 ratio across all channels into which is spiked TMTpro16-labeled yeast peptides in triplicate at 20:1, 10:1, 4:1, and 2:1 ratios. We showcase the HYpro16 standard by (1) altering the MS2 isolation window width and (2) examining different data acquisition methods (hrMS2, SPS-MS3, RTS-MS3). Our data illustrate that wider isolation widths moderately increase the TMT signal, the benefits of which are offset by decreased ratio accuracy. We also show that using real-time database searching (RTS)-MS3 resulted in the most accurate ratios. Additionally, the number of quantified yeast proteins using RTS-MS3 approaches that of hrMS2 when using a yeast-specific database for real-time searching. In short, this quality control standard allows for the assessment of multiple quantitative measurements within a single run, which can be compared across instruments to benchmark and track performance.