Informace o publikaci

Reversed-Phase Liquid Chromatography of Peptides for Bottom-Up Proteomics: A Tutorial

Autoři	LENČO Juraj JADEJA Siddharth NAPLEKOV Denis K. KROKHIN Oleg V. KHALIKOVA Maria A. NOVÁKOVÁ Petr URBAN Jiří BROECKHOVEN Ken NOVÁKOVÁ Lucie ŠVEC František
Rok publikování	2022
Druh	Článek v odborném periodiku
Časopis / Zdroj	Journal of Proteome Research
Fakulta / Pracoviště MU	Přírodovědecká fakulta
Citace
www	https://pubs.acs.org/doi/10.1021/acs.jproteome.2c00407
Doi	http://dx.doi.org/10.1021/acs.jproteome.2c00407
Klíčová slova	bottom-up proteomics; peptide separation; reversed-phase liquid chromatography; peptide properties; method development; protein separation; tutorial
Popis	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.