Phosphoproteomics by Highly Selective IMAC Protocol

Protein phosphorylation plays an important role in biological process such as cell differentiation, cell cycle control, metabolism, and apoptosis. Toward global analysis of the phosphoproteome, enrichment is an essential step to overcome analytical challenges associated with the nature of phosphoprotein, including their dynamic modification patterns, substoichiometric concentrations, heterogeneous forms of phosphoproteins, and low mass spectrometric response. Here, based on detailed evaluation of the capture and release mechanism in immobilized metal affinity chromatography (IMAC), we provide a pH/acid-controlled IMAC protocol for phosphopeptide purification with high specificity and lower sample loss. Based on a model study on non-small-cell lung cancer cell, better than 90% phosphopeptide enrichment specificity can be achieved without the use of commonly adapted methyl esterification procedure. In addition, the protocol is compatible to fractionation using SDS-PAGE. We have successfully employed the pH/acid-controlled IMAC enrichment strategy to characterize over 2,360 nondegenerate phosphopeptides and 2,747 phosphorylation sites in H1299 lung cancer cell line. We expect that the simple and reproducible IMAC protocol can be applied, fully automated or manual, for large-scale identification of the vastly under-explored phosphoproteome associated with neurodegenerative diseases.