LAUSR

Peptide separation orthogonality for 16 different 2D LC-ESI MS systems has been evaluated. To compare and contrast the behavior of the first dimension columns, a large proteomic retention dataset of ~30,000 tryptic peptides was collected for each 2D pairing. The selection of the first dimension system was made to cover the most popular peptide separation modes applied in proteomics: reversed-phase (RP) separations with different pH, hydrophilic interaction liquid chromatography (HILIC), strong cation- and anion exchange (SCX, SAX), and mixed mode separations. The separation orthogonality generally increases in the order RP < SCX < HILIC < SAX, with the exception of high pH RP - low pH RP system, which showed the second best orthogonality value (68%), just behind PolySAX LP column (74%). The identification output of 2D LC-MS/MS system is driven by both separation orthogonality and efficiency, making high pH RP the best choice for the first dimension separation. Its performance in combination with a standard C18 at acidic pH can be increased further through the application of pairwise fraction concatenation. The effect of the latter has been evaluated using in-silico fraction concatenation, which proven to show improvement only for RP separations in the first dimension. Concatenation of 2, 3 and 4-5 fractions into one is shown to be the most effective for high pH RP, HFBA- and TFA-based C18 separations, respectively. We also suggest simple guidelines for the unbiased determination of dissimilarity for two separation dimensions and evaluate separation orthogonality in 3D LC-LC-MS separation space for all systems under investigation.