Phosphopeptide enrichment and fractionation by using Click OEG-CD matrix

Abstract

Reversible protein phosphorylation regulates many significant cellular processes. Identification of phosphorylation sites is vital to elucidate their biofunctions. To aid in phosphoproteome characterization, several selective enrichment methods have been developed, including immobilized metal-ion affinity chromatography (IMAC) and titanium dioxide (TiO₂). However, the high pH elution step applied in these two methods, which tends to degrade phosphopeptides. In order to improve phosphopeptide enrichment efficiency, a hydrophilic interaction liquid chromatography (HILIC) based material, cyclodextrin (CD) bonded silica (Click OEG-CD), was synthesized in our group and applied to phosphopeptide enrichment and fractionation. Taking tryptic digest of standard protein α-casein as model sample, the performance of Click OEG-CD in phosphopeptide isolation was investigated. It was found that both the acetonitrile and salt concentrations in mobile phase influence the phosphopeptide enrichment selectivity of the matrix. Under optimized enrichment condition, Click OEG-CD has a similar phosphopeptide enrichment selectivity as commercial TiO₂. Meanwhile, phosphopeptides with same charges could be fractionated according to hydrophilicity difference on Click OEG-CD under acetonitrile gradient. Different selectivity is proved with Click OEG-CD from conventional SAX. We demonstrate that Click OEG-CD is an efficient matrix in phosphopeptide enrichment and fractionation.