Peptide Separation Techniques in Proteomics: Analysis of Peptide Separation Behavior in Reverse-Phase Chromatography and Ion Mobility Spectrometry

Abstract

In LC/MS/MS-based bottom-up proteomics, the separation and purification of peptides are crucial steps that significantly impact the identification and quantitation results. Peptide separation is influenced by various factors, necessitating the development of conditions that integrate both liquid chromatography (LC) and mass spectrometry (MS). Additionally, to maximize the utility of retention time information in LC, it is essential to accurately predict retention times under diverse conditions. Moreover, with the recent advancements in ion mobility spectroscopy, the significance of ion mobility data has grown. This paper summarizes the condition-specific separation behavior of phosphorylated peptides in reversed-phase chromatography and trapped ion mobility spectrometry (TIMS) that we have investigated and discusses how these findings can enhance sample preparation and quantitative analysis.