title: Open tubular immobilized metal ion affinity chromatography combined with MALDI MS and MS/MS for identification of protein phosphorylation sites.
abstract: Protein phosphorylation is one of the most important known posttranslational modifications. Tandem mass spectrometry has become an important tool for mapping out the phosphorylation sites. However, when a peptide generated from the enzymatic or chemical digestion of a phosphoprotein is highly phosphorylated or contains many potential phosphorylation residues, phosphorylation site assignment becomes difficult. Separation and enrichment of phosphopeptides from a digest mixture is desirable and often a critical step for MS/MS-based site determination. In this work, we present a novel open tubular immobilized metal ion affinity chromatography (OT-IMAC) method, which is found to be more effective and reproducible for phosphopeptide enrichment, compared to a commonly used commercial product, Ziptip from Millipore. A strategy based on a combination of OT-IMAC, sequential dual-enzyme digestion, and matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight tandem mass spectrometry for phosphoprotein characterization is presented. It is shown that MALDI MS/MS with collision-induced dissociation can be very effective in generating fragment ion spectra containing rich structural information, which enables the identification of phosphorylation sites even from highly phosphorylated peptides. The applicability of this method for real world applications is demonstrated in the characterization and identification of phosphorylation sites of a Na(+)/H(+) exchanger fusion protein, His182, which was phosphorylated in vitro using the kinase Erk2.
authors.affiliation: Departments of Chemistry and Biochemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
authors.forename: Huaizhi
authors.initials: H
authors.lastname: Liu
authors.forename: Jacek
authors.initials: J
authors.lastname: Stupak
authors.forename: Jing
authors.initials: J
authors.lastname: Zheng
authors.forename: Bernd O
authors.initials: BO
authors.lastname: Keller
authors.forename: Brenda J
authors.initials: BJ
authors.lastname: Brix
authors.forename: Larry
authors.initials: L
authors.lastname: Fliegel
authors.forename: Liang
authors.initials: L
authors.lastname: Li