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High-performance liquid chromatography (HPLC) is widely used for separation of complex peptide mixtures before mass spectrometry-based proteome analysis. In this analysis, reversed phase HPLC (RP-HPLC) using non-polar stationary phases such as surface-modified silica containing alkyl groups (e.g., C18) is typically employed. Because of the high heterogeneity of proteomic samples, multidimensional separation approaches gained increasing attention recently to tackle this complexity and extremely high range of concentrations. In two-dimensional liquid chromatography, hydrophilic interaction chromatography (HILIC) is often a method of choice for combination with RP-HPLC because it uses reversed-phase type eluents and allows efficient separation of polar peptides. Due to the high degree of orthogonality in this two-dimensional separation space, it is tempting to develop approaches for predicting peptide retention times for HILIC-based separations similar to the ones for RP-HPLC. Recent successful efforts in this area were focused on developing retention coefficient (RC)-based approaches. Herein, we explored the feasibility of using a statistical thermodynamic model for prediction of peptide retention times in HILIC separations and determined the phenomenological parameters of the model for a bare silica column. The performance of the developed model was tested using HPLC-MS analysis of a set of synthetic peptides, as well as a tryptic peptide mixture. [ABSTRACT FROM AUTHOR]

State-of-the art two-dimensional (2-D) nano LC coupled to mass spectrometry (MS) provides a very powerful analytical tool for the separation, identification and characterization of complex proteomics samples such as human tissue. This approach is usually based on the injection of the digested protein sample onto a strong cation exchange (SCX) column as first dimension of the 2-D separation. A problem with this approach, however, is that injecting salt plugs onto the SCX column limits the separation power, resulting in lower chromatographic resolution. To overcome these shortcomings, LC Packings/Dionex has developed the UitiMateTM dual gradient nano LC system.