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

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).