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 such as cell lysates. One reason is the reduced sensitivity of the methods, with second separation dimensions that run at high flow rates, resulting in the need for flow splitters to couple to MS and inducing high dilution factors. This study reports proof of principle results of the development of a LC×LC-HRMS using parallel gradients for the analysis of complex digests, removing the need for post-column splitting pre-MS with reduced flow rates in the second dimension (0.7 mL∙min-1). With this strategy, high 2D surface coverage and peak capacity were obtained (peak capacity of 679 in 60 minutes). The analysis of human cell culture lysate digest by parallel RPLC×RPLC MS/MS resulted in the identification of 8959 peptides and 1984 proteins within 1hr run. This was a gain of 149% in the number of peptides identified compared to 1D-LC method. Parameters such as the gradient program, flow rate and modulation time were investigated. This approach reduces the complexity of gradient programming as two simple linear gradients can be programmed in both dimensions, eliminating the need for column re-equilibration between different modulations, making it attractive for LC×LC methods at low flowrates. Prospectives on the limits and application of such methods are discussed.