Parallel Notched Gas-Phase Enrichment for Improved Proteome Identification and Quantification with Fast Spectral Acquisition Rates.

Gas-phase fractionation enables better quantitative accuracy, improves signal-to-noise ratios, and increases sensitivity in proteomic analyses. However, traditional gas-phase enrichment, which relies upon a large continuous bin, results in suboptimal enrichment, as most chromatographic separations are not 100% orthogonal relative to the first MS dimension (MS/). As such, ions with similar / values tend to elute at the same retention time, which prevents the partitioning of narrow precursor / distributions into a few large continuous gas-phase enrichment bins. To overcome this issue, we developed and tested the use of notched isolation waveforms, which simultaneously isolate multiple discrete / windows in parallel (e.g., 650-700 / and 800-850 /). By comparison to a canonical gas-phase fractionation method, notched waveforms do not require bin optimization via digestion or wasteful sample injections to isolate multiple precursor windows. Importantly, the collection of all / bins simultaneously using the isolation waveform does not suffer from the sensitivity and duty cycle pitfalls inherent to sequential collection of multiple / bins. Applying a notched injection waveform provided consistent enrichment of precursor ions, which resulted in improved proteome depth with greater coverage of low-abundance proteins. Finally, using a reductive dimethyl labeling approach, we show that notched isolation waveforms increase the number of quantified peptides with improved accuracy and precision across a wider dynamic range.