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Specific O‐GlcNAcylated Peptide Enrichment with Improved Photocleavable Chemical/Enzymatic Tagging Methodology
Author(s) -
Han Guanghui,
Ma Junfeng,
Liu Xin,
Hart Gerald W.
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.776.7
Subject(s) - chemistry , threonine , biotinylation , serine , chromatography , peptide , reagent , phosphorylation , bovine serum albumin , biochemistry , enzyme , cleavage (geology) , mass spectrometry , biology , paleontology , fracture (geology)
Like phosphorylation, the addition of O‐linked beta‐N‐acetylglucosamine (O‐GlcNAcylation) which modifies serine and threonine residues on nuclear and cytoplasmic proteins is a ubiquitous, reversible process that regulates numerous cellular processes. Although several methods exist for the enrichment and identification of protein O‐GlcNAcylation sites, developing more sensitive enrichment methods are advantageous. Previously, we reported one very powerful methodology for the enrichment and characterization of O‐GlcNAc sites from complex samples: O‐GlcNAcylated peptides are tagged with a biotinylation reagent, enriched by affinity chromatography, and then released from the solid support by photochemical cleavage. Here, an improved method was reported by optimizing reaction conditions and more sensitivity was achieved by using less enrichment steps. The sensitivity of the improved method was demonstrated by selective enrichment of O‐GlcNAcylated peptides from mixture of tryptic digests of alpha‐crystalline and bovine serum albumin (BSA) at ratio of 1 to 1000 by LC‐MS/MS analysis. We are further applying this improved method to identify the O‐GlcNAcylation sites from proteins in prostate cancer cells.