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 methods' reduced sensitivity that 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 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 a 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 an effective peak capacity of 779 and 757 in 60 minutes). 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 10s 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 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).