Poster Presentation 29th Annual Lorne Proteomics Symposium 2024

Real-Time Search improves sensitivity of TMTpro complementary ion quantification (#196)

Alex Johnson 1 , Rafael Melani 2 , Jingjing Huang 2 , William Barshop 2 , Jesse Canterbury 2 , Amanda Lee 2 , Vlad Zabrouskov 2 , Graeme McAlister 2 , Martin Wühr 1
  1. Princeton University, Princeton, United States
  2. Thermo Fisher Scientific, San Jose, CALIFORNIA, United States

Using the balancer-peptide conjugates (TMTproC complementary ions) in the MS2 spectra for quantification circumvents the ratio distortion problem of multiplexed proteomics. These TMTproC quantification scans require long transient and ion injection times for sufficient ion statistics and spectral resolution. Real-Time Search (RTS) algorithms have been shown to increase the sensitivity of SPS-MS3 methods. By informed selection of precursor peaks for quantification, analysis time can be prioritized to peptides useful for quantifying proteins. Nevertheless, the naive implementation of TMTproC still quantifies more proteins than SPS-MS3. Here, we combine complementary ion quantification with Real-Time Search (TMTproC-RTS) to improve sensitivity while maintaining accuracy and precision in quantitative proteomics experiments at the MS2 level. Human peptides were labeled with TMTpro at 1:1 ratios across the nine 1-Da spaced channels in the complementary ion region. Similarly, yeast peptides were labeled in ratios of 0:1:5:10:1:10:5:1:0 with TMTpro. Human and yeast peptides were mixed at a ratio of 10:1 before analysis, and the mixed proteome was analyzed on an Orbitrap Ascend Tribrid. The TMTproC-RTS method collects fast exploratory ion trap MS2 scans at the turbo scan rate with quadrupole isolation and HCD fragmentation. MS2 spectra are searched immediately against a concatenated human-yeast fasta file. Successfully identified peptides were then re-isolated, and high-resolution MS2 spectra were collected in the Orbitrap with quadrupole isolation and CID fragmentation. In addition to ppm, XCorr, and dCn filters, in separate runs, the RTS filter was set to trigger a high-res MS2 scan on either peptides originating from yeast proteins, nuclear proteins, ortranscription factors. We find that TMTproC-RTS increases protein identifications by 10-100% depending on the proportion of the proteome that is included in the RTS target list. Thus, TMTproC-RTS enables the quantification of low-abundant proteins of interest, like transcription factors and signaling molecules, in multiplexed experiments.