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 >10x increase in precursor isolation specificity as compared to either of the techniques independently.