More than 100,000 Detectable Peptide Species Elute in Single Shotgun Proteomics Runs but the Majority is Inaccessible to Data-Dependent LC−MS/MS

TLDR

Shotgun proteomics aims to identify as many peptides as possible from complex mixtures, but the proportion of additional peptides that can be identified is limited by sequencing speed, sensitivity, and precursor ion isolation. The study investigates how many peptides are detectable by high‑resolution MS in standard LC runs of cell lysate and how many of those are accessible to data‑dependent MS/MS. The authors used MaxQuant to determine isotope clusters and stringently filtered them for typical peptide charge states and retention times. The analysis revealed over 100,000 peptide features, yet only ~16% were targeted for MS/MS; achieving full coverage would require a 25 s⁻¹ scan rate, but typical rates fall below 17 s⁻¹ for abundant peptides, with sensitivity demanding long injection times (>250 ms) and a median precursor ion fraction of only 0.14, highlighting key limitations for increasing shotgun proteomics coverage.

Abstract

Shotgun proteomics entails the identification of as many peptides as possible from complex mixtures. Here we investigate how many peptides are detectable by high resolution MS in standard LC runs of cell lysate and how many of them are accessible to data-dependent MS/MS. Isotope clusters were determined by MaxQuant and stringently filtered for charge states and retention times typical of peptides. This resulted in more than 100 000 likely peptide features, of which only about 16% had been targeted for MS/MS. Three instrumental attributes determine the proportion of additional peptides that can be identified: sequencing speed, sensitivity, and precursor ion isolation. In our data, an MS/MS scan rate of 25/s would be necessary to target all peptide features, but this drops to less than 17/s for reasonably abundant peptides. Sensitivity is a greater challenge, with many peptide features requiring long MS/MS injection times (>250 ms). The greatest limitation, however, is the generally low proportion of the target peptide ion intensity in the MS/MS selection window (the "precursor ion fraction" or PIF). Median PIF is only 0.14, making the peptides difficult to identify by standard MS/MS methods. Our results aid in developing strategies to further increase coverage in shotgun proteomics.