Department of Proteomics and Bioanalytics, Center of Life and Food Sciences Weihenstephan, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany.
J Am Soc Mass Spectrom. 2011 May;22(5):931-42. doi: 10.1007/s13361-011-0107-y. Epub 2011 Mar 26.
The modification of serine and threonine residues in proteins by a single N-acetylglucosamine (O-GlcNAc) residue is an emerging post-translational modification (PTM) with broad biological implications. However, the systematic or large-scale analysis of this PTM is hampered by several factors, including low stoichiometry and the lability of the O-glycosidic bond during tandem mass spectrometry. Using a library of 72 synthetic glycopeptides, we developed a two-stage tandem MS approach consisting of pulsed Q dissociation (PQD) for O-GlcNAc peptide detection and electron transfer dissociation (ETD) for identification and site localization. Based on a set of O-GlcNAc specific fragment ions, we further developed a score (OScore) that discriminates O-GlcNAc peptide spectra from spectra of unmodified peptides with 95% sensitivity and >99% specificity. Integrating the OScore into the two-stage LC-MS/MS approach detected O-GlcNAc peptides in the low fmol range and at 10-fold better sensitivity than a single data-dependent ETD tandem MS experiment.
蛋白质中丝氨酸和苏氨酸残基被单个 N-乙酰葡萄糖胺（O-GlcNAc）残基修饰是一种新兴的翻译后修饰（PTM），具有广泛的生物学意义。然而，由于几个因素的限制，包括低化学计量和糖肽在串联质谱中 O-糖苷键的不稳定性，对这种 PTM 的系统或大规模分析受到了阻碍。利用 72 种合成糖肽文库，我们开发了一种两阶段串联 MS 方法，包括用于 O-GlcNAc 肽检测的脉冲 Q 解离（PQD）和用于鉴定和位点定位的电子转移解离（ETD）。基于一组 O-GlcNAc 特异性片段离子，我们进一步开发了一种评分（OScore），可将 O-GlcNAc 肽谱与未经修饰的肽谱区分开来，具有 95%的灵敏度和>99%的特异性。将 OScore 整合到两阶段 LC-MS/MS 方法中，可以在低 fmol 范围内检测到 O-GlcNAc 肽，比单个基于数据的 ETD 串联 MS 实验的灵敏度提高了 10 倍。
