High‐Resolution Ion Mobility as an Alternative to Quadrupole‐Based Precursor Isolation for Reducing Chimeric Fragmentation Spectra in Bottom‐Up Proteomics

To assess the potential for high resolution ion mobility (HRIM) as an alternative means of precursor isolation for mass spectrometry fragmentation analysis we performed a meta-analysis of predicted tryptic peptide features from the human proteome to measure the rate of chimeric spectrum generation relative to traditional quadrupole-based isolation. Results indicate that for proteomic mixtures, HRIM separation with a peak capacity of 100 produces chimeric spectra at a rate commensurate with a ∼5 Th quadrupole isolation window, while providing the additional benefit of generating non-chimeric spectra for many isobaric and isomeric peptides unresolvable with a quadrupole filter. This behavior was verified experimentally using a HRIM-QTOF mass spectrometry system. The ability to combine HRIM and MS isolation resulted in >10× increase in precursor isolation specificity as compared to either of the techniques independently.