ABSTRACT
Glycosylation and phosphorylation represent the majority of posttranslational protein modi“cations and it is widely accepted that they are substantially involved in numerous essential cellular processes such as inter-and intracellular signaling, metabolism, protein synthesis and degradation, and cell survival. For this reason, functional glycomics, glycoproteomics, and phosphoproteomics are rapidly growing research areas. One of the most prominent challenges associated with these disciplines is the substoichiometric presence of these modi“cations, resulting from substantial heterogeneity of glycosylation as well as frequently a low degree of phosphorylation of a given site. Fractionation or enrichment of the modi“ed proteins is consequently essential to alleviate this problem. However, the signi“cant hydrophilicities associated with these biomolecules limit the use of traditional puri“cation techniques such as reversed-phase liquid chromatography (RPLC) setups. In contrast, hydrophilic interaction chromatography (HILIC), which features the opposite characteristics of RPLC, is an attractive technique for the analysis of hydrophilic compounds and applications usually involve detection by mass spectrometry (MS) to bene“t from its high sensitivity, accuracy, and resolution as well as its high throughput potential.
