Comprehensive Two-Dimensional Liquid Chromatography-High-Resolution Mass Spectrometry for Complex Protein Digest Analysis Using Parallel Gradients

Abstract

Despite the high gain in peak capacity, online comprehensive two-dimensional liquid chromatography coupled with high-resolution mass spectrometry (LC × LC-HRMS) has not yet been widely applied to the analysis of complex protein digests. One reason is the method's reduced sensitivity which can be linked to the high flow rates of the second separation dimension (²D). This results in higher dilution factors and the need for flow splitters to couple to ESI-MS. This study reports proof-of-principle results of the development of an RPLC × RPLC-HRMS method using parallel gradients (²D flow rate of 0.7 mL min^-1) and its comparison to shifted gradient methods (²D of 1.4 mL min^-1) for the analysis of complex digests using HRMS (QExactive-Plus MS). Shifted and parallel gradients resulted in high surface coverage (SC) and effective peak capacity (SC of 0.6226 and 0.7439 and effective peak capacity of 779 and 757 in 60 min). When applied to a cell line digest sample, parallel gradients allowed higher sensitivity (e.g., average MS intensity increased by a factor of 3), allowing for a higher number of identifications (e.g., about 2600 vs 3900 peptides). In addition, reducing the modulation time to 10 s significantly increased the number of MS/MS events that could be performed. When compared to a 1D-RPLC method, parallel RPLC × RPLC-HRMS methods offered a higher separation performance (FHWH from 0.12 to 0.018 min) with limited sensitivity losses resulting in an increase of analyte identifications (e.g., about 6000 vs 7000 peptides and 1500 vs 1990 proteins).

Figure 1

Orthogonality plots using normalized retention times (ntr) of targeted peptide features. The following comparisons are presented: C18 using 0.1% FA (x-axis in all subplots) vs HPH C18 using 20 mM AmFm at pH 10 (A), cyano using 0.1% FA (B).

Figure 2

Two-dimensional LC plots of the BPC obtained from the protein-mixture digest sample using different gradient assemblies: full gradient (A), shifted gradient (B), and parallel gradient (C). In all plots, the intensity is represented by color and scaled to a relative intensity such that all chromatograms appear equally visible despite absolute differences in peak heights. It should be noted for ease of visibility, the ²D times in Figure A, B have been shifted by 0.07 min and C by 0.24 min to account for dead time.

Figure 3

Base-peak RPLC × RPLC-MS/MS chromatograms of a cell lystate of human IMR90 lung fibroblast cells using the shifted-gradient method (A) and the parallel-gradient method using 30 s modulations (B) and 10 s modulations (C). 1DLC analysis view of the data can be found in Figure S11.