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

• Published in: Journal of proteome research Vol. 19; no. 7; pp. 2750 - 2757
• Main Authors: Erickson, Brian K, Schweppe, Devin K, Yu, Qing, Rad, Ramin, Haas, Wilhem, McAlister, Graeme C, Gygi, Steven P
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 02.07.2020
• Subjects: chromatography; computer simulation; fractionation; gases; ions; peptides; proteins; proteome; proteomics; signal-to-noise ratio; gas-phase fractionation; injection waveform; multinotch; orbitrap; label-free quantification; reductive dimethylation; dynamic range
• ISSN: 1535-3893; 1535-3907; 1535-3907
• DOI: 10.1021/acs.jproteome.9b00715

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 (MS1 m/z). As such, ions with similar m/z values tend to elute at the same retention time, which prevents the partitioning of narrow precursor m/z 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 m/z windows in parallel (e.g., 650–700 m/z and 800–850 m/z). By comparison to a canonical gas-phase fractionation method, notched waveforms do not require bin optimization via in silico digestion or wasteful sample injections to isolate multiple precursor windows. Importantly, the collection of all m/z bins simultaneously using the isolation waveform does not suffer from the sensitivity and duty cycle pitfalls inherent to sequential collection of multiple m/z 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.