biostudies-literatureUnknown6(1)Agrawal NCell signalling is a vital process in cell physiology, which is driven by protein phosphorylation. Global phosphoproteome analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has thus gained importance in cell signalling research. However, phosphoprotein identification by LC-MS/MS in whole cell lysates, which are complex protein mixtures, is hindered by their poor ionization coupled with suppression of peaks due to low abundance. Enrichment by immobilized metal ion- and metal oxide-affinity chromatography (IMAC and MOAC), which preferentially enrich multi- and mono-phosphorylated proteins, respectively, have improved their detection by MS. However, preferential enrichment limits phosphoproteome coverage in global analyses of cell lysates which contain mono- and multi-phospAnalytical science advancese202400058https://www.ebi.ac.uk/biostudies/studies/S-EPMC11789765biostudies-literatureIncremental Modification in the Existing Approaches for Affinity Chromatographic Enrichment of Phosphoproteins Improves Their Profile in Liquid Chromatography-Tandem Mass Spectrometry Analysis.PMC11789765Govekar RAgrawal NfalseIncremental Modification in the Existing Approaches for Affinity Chromatographic Enrichment of Phosphoproteins Improves Their Profile in Liquid Chromatography-Tandem Mass Spectrometry Analysis.Cell signalling is a vital process in cell physiology, which is driven by protein phosphorylation. Global phosphoproteome analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has thus gained importance in cell signalling research. However, phosphoprotein identification by LC-MS/MS in whole cell lysates, which are complex protein mixtures, is hindered by their poor ionization coupled with suppression of peaks due to low abundance. Enrichment by immobilized metal ion- and metal oxide-affinity chromatography (IMAC and MOAC), which preferentially enrich multi- and mono-phosphorylated proteins, respectively, have improved their detection by MS. However, preferential enrichment limits phosphoproteome coverage in global analyses of cell lysates which contain mono- and multi-phosp2025-01-01T00:00:00Z2025 Jun2026-06-02T04:23:38.351Z2025-04-19T16:24:44.419ZS-EPMC117897653990246410.1002/ansa.202400058