Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection.
The diversity and complexity of proteins and peptides in biological systems requires powerful liquid chromatography-based separations to optimize resolution and detection of components. Proteomics strategies often combine two orthogonal separation modes to meet this challenge. In nearly all cases, the second dimension is a reverse phase separation interfaced directly to a mass spectrometer. Here we report on the use of hydrophilic interaction chromatography (HILIC) as part of a multidimensional chromatography strategy for proteomics. Tryptic peptides are separated on TSKgel Amide-80 columns using a shallow inverse organic gradient. Under these conditions, peptide retention is based on overall hydrophilicity, and a separation truly orthogonal to reverse phase is produced. Analysis of tryptic digests from HeLa cells yielded numbers of protein identifications comparable to that obtained using strong cation exchange. We also demonstrate that HILIC represents a significant advance in phosphoproteomics analysis. We exploited the strong hydrophilicity of the phosphate group to selectively enrich and fractionate phosphopeptides based on their increased retention under HILIC conditions. Subsequent IMAC enrichment of phosphopeptides from HILIC fractions showed better than 99% selectivity. This was achieved without the use of derivatization or chemical modifiers. In a 300-microg equivalent of HeLa cell lysate we identified over 1000 unique phosphorylation sites. More than 700 novel sites were added to the HeLa phosphoproteome.
McNulty
Dean E
DE
Proteomics and Biological Mass Spectrometry Laboratory, GlaxoSmithKline, King of Prussia, Pennsylvania 19406, USA.
Annan
Roland S
RS
D002853:Chromatography, Liquid / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D010748:Phosphopeptides / Q000032:analysis / Q000302:isolation & purification*; D020543:Proteome / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*
D010748:Phosphopeptides; D020543:Proteome