MassIVE MSV000092199 - Parallel gradients in comprehensive two_dimensional liquid chromatography_mass spectrometry for complex protein digest analysis

Here we describe the development of a parallel gradient method for protein digest analysis, investigating parameters such as gradient program, flow rate and modulation time. High 2D surface coverage and peak capacity were obtained (peak capacity of 679 in 60 minutes). Moreover, the use of parallel gradients is favorable to favor LCxLC HRMS coupling, removing the need for postcolumn splitting preHRMS with allowing separation at reduced flow rates in the second dimension (0.7 mL/ min), removing the need for post-column splitting pre-HRMS. The analysis of human cell culture lysate digest by parallel RPLCxRPLC MS/MS resulted in identifying 8959 peptides and 1984 proteins within 1hr run. This was a gain of 149% in the number of peptides identified compared to 1DLC method.