METHOD FOR ISOLATING AND IDENTIFYING O-GLcNAc PEPTIDE USING METAL-AFFINITY CHROMATOGRAPHY

摘要

PURPOSE: A method for isolating and identifying O-GlcNAc peptide using metal-affinity chromatography is provided to quickly and easily isolate O-GlcNAc peptide from phosphorylated peptide without chemical modification, and to identify the peptide sequence and modification site from the isolated O-GlcNAc peptide.;CONSTITUTION: A method for isolating and identifying O-GlcNAc peptide comprises: a step of performing metal-affinity chromatography of a sample containing a phosphorylated peptide and O-GlcNAc peptide; and a step of adsorbing and removing the phosphorylated peptide onto a column. Metals used for metal affinity chromatography are TiO_2, ZrO_2, GaO_4, or FeO_2. The method also comprises: a step of adding a dithiothreitol(DTT) affinity tag to O-GlcNAc peptide in the sample by BEMAD method; and a step of purifying O-GlcNAc peptide using thiol affinity chromatography. The method further comprises a step of analyzing O-GlcNAc peptide by LC-MS(liquid chromatography-mass spectrometry).;COPYRIGHT KIPO 2013;[Reference numerals] (AA) Target sample to be analyzed; (BB) Metal affinity chromatography; (CC) O-GlcNac peptide containing sample; (DD) BEMAD method; (EE) DTT added peptide; (FF) Thiol chromatography, LC-MS/MS mass spectrometry; (GG) O-GlcNac peptide