Separation Orthogonality in Liquid Chromatography-Mass Spectrometry for Proteomic Applications: Comparison of 16 Different Two-Dimensional Combinations.

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 data set 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 the 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 has been proven to show improvement only for RP separations in the first dimension. Concatenation of two, three, and four-five fractions into one is shown to be the most effective for high pH RP and 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.