The performance of the current bottom-up liquid chromatography hyphenated with mass spectrometry (LC-MS) analyses has undoubtedly been fueled by spectacular progress in mass spectrometry. It is thus not surprising that the MS instrument attracts the most attention during LC-MS method development, whereas optimizing conditions for peptide separation using reversed-phase liquid chromatography (RPLC) remains somewhat in its shadow. Consequently, the wisdom of the fundaments of chromatography is slowly vanishing from some laboratories. However, the full potential of advanced MS instruments cannot be achieved without highly efficient RPLC. This is impossible to attain without understanding fundamental processes in the chromatographic system and the properties of peptides important for their chromatographic behavior. We wrote this tutorial intending to give practitioners an overview of critical aspects of peptide separation using RPLC to facilitate setting the LC parameters so that they can leverage the full capabilities of their MS instruments. After briefly introducing the gradient separation of peptides, we discuss their properties that affect the quality of LC-MS chromatograms the most. Next, we address the in-column and extra-column broadening. The last section is devoted to key parameters of LC-MS methods. We also extracted trends in practice from recent bottom-up proteomics studies and correlated them with the current knowledge on peptide RPLC separation.

Department of Analytical Chemistry Faculty of Pharmacy in Hradec Králové Charles University Heyrovského 1203 8 500 05Hradec Králové Czech Republic

Department of Chemical Engineering Faculty of Engineering Vrije Universiteit Brussel Pleinlaan 2 1050Brussel Belgium

Department of Chemistry Faculty of Science Masaryk University Kamenice 5 625 00Brno Czech Republic

Department of Internal Medicine Manitoba Centre for Proteomics and Systems Biology University of Manitoba 799 JBRC 715 McDermot Avenue WinnipegR3E 3P4 Manitoba Canada

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