Systematic characterization of site-specific proline hydroxylation using hydrophilic interaction chromatography and mass spectrometry (RCC4)

To improve understanding of the role of site-specific proline hydroxylation in controlling protein function, we have developed a robust workflow for the identification of proline hydroxylation sites in proteins using a combination of hydrophilic interaction chromatography (HILIC) enrichment and high-resolution nano-Liquid Chromatography-Mass Spectrometry (LC-MS). Using this approach, together with refining and filtering parameters during data analysis, by combining the results from cell lines being treated with either the prolyl hydroxylase inhibitor Roxadustat (FG-4592, FG) or the proteasome inhibitor MG-132 (MG), or DMSO, a total of 4,993 and 3,247 proline hydroxylation sites were identified in HEK293 (PT10822) and RCC4 (PT10402) samples.