Comparison of Protein Quantification in a Complex Background by DIA and TMT Workflows with Fixed Instrument Time

Label-free quantification (LFQ) and isobaric labeling quantification (ILQ) are among the most popular protein quantification workflows in discovery proteomics. Here, we compared the TMT SPS/MS3 10-plex workflow to a label free single shot data-independent acquisition (DIA) workflow on a controlled sample set. The sample set consisted of ten samples derived from 10 biological replicates of mouse cerebelli spiked with the UPS2 protein standard in five different concentrations. For a fair comparison, we matched the instrument time for the two workflows. The LC–MS data were acquired at two facilities to assess interlaboratory reproducibility. Both methods resulted in a high proteome coverage (>5000 proteins) with low missing values on protein level (<2%). The TMT workflow led to 15–20% more identified proteins and a slightly better quantitative precision, whereas the quantitative accuracy was better for the DIA method. The quantitative performance was benchmarked by the number of true positives (UPS2 proteins) within the top 100 candidates. TMT and DIA showed a similar performance. The quantitative performance of the DIA data stayed in a similar range when searching the spectra against a fasta database directly, instead of using a project-specific library. Our experiments also demonstrated that both workflows are readily transferrable between facilities.