A Multidimensional Chromatography Strategy Using HILIC and IMAC for Quantitative Phosphoproteome Analysis

Abstract

Mass spectrometry-based protein phosphorylation analysis on a proteome-wide scale remains a formidable challenge, hampered by the complexity and dynamic range of protein expression on the global level and multi-site phosphorylation at substoichiometric ratios at the individual protein level. Reduction of sample complexity and enrichment of the phosphopeptide pool is a necessary prerequisite for global phosphoproteomics. Metal affinity chromatography and strong cation exchange chromatography, either alone or in tandem, have emerged as the most widely used chromatographic and enrichment strategies. However, each is not without shortcomings. The former suffers from selectivity over highly acidic peptides, while the latter is a low resolution technique that provides little in the way of enrichment for phosphorylated peptides. Here we describe a phosphopeptide fractionation scheme using hydrophilic interaction chromatography (HILIC) which both enriches the phosphopeptide pool and efficiently fractionates the remaining peptides. When HILIC is used in front of metal affinity chromatography, the selectivity of the metal affinity resin is improved to greater than 90%. The lack of significant numbers of nonphosphorylated peptides also allows for more efficient use of the mass spectrometer duty cycle in that the instrument spends nearly all of its time sequencing phosphopeptides.