Multiplexed proteomics is a powerful tool to assay cell states in health and disease, but accurate quantification of relative protein changes is impaired by interference from co-isolated peptides. Interference can be reduced by using MS3-based quantification, but this reduces sensitivity and requires specialized instrumentation. An alternative approach is quantification by complementary ions, which allows accurate and precise multiplexed quantification at the MS2 level and is compatible with the most widely distributed instruments. However, complementary ions of the popular TMT tag form inefficiently and multiplexing is limited to five channels. Here, we evaluate and optimize complementary ion quantification for the recently released TMTPro tag, which increases plexing capacity to eight channels (TMTProC). We find that the beneficial fragmentation properties of TMTPro increase quantification signal five-fold compared to TMT. This increased sensitivity results in ~65% more proteins quantified compared to TMTPro-MS3 and even slightly outperforms TMTPro-MS2. Furthermore, TMTProC quantification is more accurate than TMTPro-MS2 and even superior to TMTPro-MS3. To demonstrate the power of TMTProC, we analyzed a human and yeast interference sample and were able to quantify 13,290 proteins in 24 fractions. Thus, TMTProC advances multiplexed proteomics data quality and widens access to accurate multiplexed proteomics beyond laboratories with MS3-capable instrumentation.

TMTPro Complementary Ion Quantification Increases Plexing and Sensitivity for Accurate Multiplexed Proteomics at the MS2 Level