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

Abstract

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 (MS¹m/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.

Keywords: dynamic range; gas-phase fractionation; injection waveform; label-free quantification; multinotch; orbitrap; reductive dimethylation.

Figure 1.

(A) Standard gas-phase fractionation utilizes continuous bins that are often optimized via in silico prediction of peptide distributions or through extra injections of the sample. The mass-dependent elution of peptides during liquid chromatographic (LC) separation limits the degree of enrichment observed with continuous bins. Each bin (red, blue, and green) is employed within an independent, replicate sample injection. (B) A series of notches in an injection waveform results in discrete m/z slices or bins across the mass range. This approach is not reliant on in silico prediction or hindered by LC peptide elution. (C) The notched injection waveform is illustrated through discrete wells in the mass range permitting the retention of ions within wells and ejection of all other ions. (D) Example spectra from an LC-MS analysis of yeast whole-cell lysate. Consecutive MS¹ spectrum were collected via a standard full mass method, a binned GPF method, and a notched injection waveform.

Figure 2.

Yeast whole-cell lysate was analyzed via LC-MS. Back-to-back full scan MS¹ spectra were collected utilizing full mass range MS¹ scan, a standard GPF scan with a large continuous bin, and an MS¹ scan with a notched GPF. Features from the standard MS¹ scan were identified in the GPF spectra, and the degree of enrichment was calculated based on the reported signal-to-noise values for the feature in each spectra. (A) Continuous (Standard GPF) and discrete (Notched GPF) were compared. In each GPF scan, the total m/z space within the mass range stayed the same. (B) The mean fold enrichment was plotted against the chromatographic retention time. The notched injection waveform exhibits more uniform enrichment when compared to the standard GPF.

Figure 3.

Method comparison for enhanced proteome characterization. (A) Yeast whole-cell lysate (YWCL) was injected three times per method. For the GPF experiments (notched, tiling, and binned), the mass range for each of the injections was shifted to permit full coverage of the entire mass range. The standard experiment utilized triplicate injections of YWCL and collected MS¹ spectra across the full mass range. The large number of MS² for the standard method reflects the redundant sampling of peptides even at a dynamic exclusion of 55 ms. The notched and standard methods resulted in the largest numbers of unique peptides. However, due to the redundant sampling of peptides in the standard method, the total number of proteins is higher in each of the GPF experiments. (B) To assess the ability of the notched method to identify low-abundance yeast proteins, the unique peptides from the standard and notched method were compared. Low copy number yeast proteins (x-axis), which were identified in both methods, are plotted according to the expected number of molecules per cell (blue-dashed line, y-axis). For each protein, the number of unique peptides from the standard method was subtracted from the number of unique peptides from the notched method, and the difference was plotted (y-axis, black bars represent more protein coverage in the standard method and red bars represent more protein coverage in the notched method). Utilizing the notched injection waveforms resulted in greater sampling of unique peptides from low-abundance yeast proteins.

Figure 4.

Yeast whole-cell lysate was split and labeled via reductive dimethylation. Light- and heavy-labeled peptides were mixed in 1:1, 5:1, 10:1, and 20:1 ratios and then analyzed through back-to-back collection of MS¹ via a standard method and by application of a notched injection waveform. For each mixture, the distribution of calculated ratios is plotted. The notched approach (red line) yielded more accurately quantified peptides at ratios of 5:1, 10:1, and 20:1.

Figure 5.

(A–C) Back-to-back comparisons of extracted ion chromatograms for peptides identified from yeast whole-cell lysate. For each mixture, a standard MS¹ and a notched MS¹ were collected consecutively. For the 20:1 sample (A), the notched method routinely resulted in higher signal-to-noise values and more accurate quantification. Across all ratios, the gain in signal-to-noise values from the notched method results in more accurate ratio quantification. (D) The notched method permits greater sampling of peptides across their elution, as was observed for the mean number of measurements across the peptide elution.