title: Hydrophilic interaction chromatography for fractionation and enrichment of the phosphoproteome.
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. It is recognized that reduction of sample complexity or enrichment of the phosphopeptide pool is a necessary prerequisite for global phospho-proteomics. Immobilized metal affinity chromatography (IMAC) and strong cation exchange chromatography, either alone or in tandem, have emerged as the most widely used chromatographic-based enrichment strategies. However, each is not without shortcomings. Both techniques provide little fractionation of phosphorylated species and are compromised by competition and co-elution of highly acidic peptides. Here, we describe a phosphopeptide prefractionation scheme using hydrophilic interaction chromatography, which both enriches the phosphopeptide pool and efficiently fractionates the remaining peptides. When used in front of IMAC, the selectivity of the metal affinity resin is improved to greater than 95%. 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 in sequencing the phosphopeptides.
authors.affiliation: Proteomics and Biological Mass Spectrometry Laboratory, GlaxoSmithKline, King of Prussia, PA, USA.
authors.forename: Dean E
authors.initials: DE
authors.lastname: McNulty
authors.forename: Roland S
authors.initials: RS
authors.lastname: Annan
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D006860:Hydrogen Bonding; D010750:Phosphoproteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification*; D020543:Proteome / Q000737:chemistry* / Q000302:isolation & purification*; D014867:Water / Q000737:chemistry / Q000494:pharmacology
chemical_list: D010750:Phosphoproteins; D020543:Proteome; D014867:Water