Isolation and Identification of Phosphopeptides by Combining Strong Cationic Exchange and Hydrophilic Interaction Chromatography

摘要

Phosphorylation is one of the most common of the reversible posttranslational modifications, but also one of the most challenging to measure. During the last few years, phosphopeptide analysis has benefited from the rapid improvement in biological mass spectrometry but also from innovations such as immobilized metal affinity chromatography (IMAC), the development of antibodies specific for phosphorylated amino acids, and most recently, chromatography using titanium oxide (TiO_(2)) [1]. During our analysis of the parotid saliva phosphoproteome, we were concerned that there is a bias against monophosphorylated peptides when using TiO_(2) columns. We therefore decided to explore alternatives that rely on fractionation rather than affinity purification.