Haynes P A, Aebersold R
Department of Molecular Biotechnology, University of Washington, Seattle 98195, USA.
Anal Chem. 2000 Nov 1;72(21):5402-10. doi: 10.1021/ac000512w.
Glycoproteins carrying O-linked N-acetylglucosamine (O-GlcNAc) modifications have been isolated from a wide range of organisms ranging from trypanosomes to humans. Interest in this modification is increasing as evidence accumulates that it is an abundant and transient modification that is dynamic and responsive to cellular stimuli. Concurrent advances in biological mass spectrometry (MS) have facilitated high-sensitivity protein identification by tandem MS. In this study, we show that the lability of the O-GlcNAc moiety to low-energy collision in tandem MS offers a means of distinguishing such peptides from others that are not modified. The differential between the energy required to remove the O-GlcNAc group and the energy required to fragment the peptide chain allows the O-GlcNAc group to be detected and the peptide sequence, and therefore the protein, to be identified. This technique thus allows the simultaneous detection and identification of O-GlcNAc-modified peptides, even when present at low levels in complex mixtures. The method was initially developed and validated using a synthetic O-GlcNAc-modified peptide and then applied to the detection of an extremely low abundance O-GlcNAc-modified peptide from bovine alpha-crystallin. We believe that with further development this assay system may prove to be a useful tool for the direct investigation of intracellular O-GlcNAc levels, thus providing valuable insights into the physiological role of O-GlcNAc modified proteins.
携带O-连接的N-乙酰葡糖胺（O-GlcNAc）修饰的糖蛋白已从从锥虫到人类的广泛生物体中分离出来。随着越来越多的证据表明这种修饰是一种丰富且短暂的修饰，具有动态性并对细胞刺激有反应，人们对这种修饰的兴趣与日俱增。生物质谱（MS）技术的同步发展促进了通过串联质谱进行高灵敏度蛋白质鉴定。在本研究中，我们表明，O-GlcNAc部分在串联质谱中对低能量碰撞的不稳定性提供了一种将此类肽与未修饰的其他肽区分开来的方法。去除O-GlcNAc基团所需的能量与肽链断裂所需的能量之间的差异使得能够检测到O-GlcNAc基团，并鉴定肽序列，进而鉴定蛋白质。因此，即使在复杂混合物中含量很低时，该技术也能同时检测和鉴定O-GlcNAc修饰的肽。该方法最初使用合成的O-GlcNAc修饰肽进行开发和验证，然后应用于检测牛α-晶状体蛋白中极低丰度的O-GlcNAc修饰肽。我们相信，随着进一步发展，该检测系统可能被证明是直接研究细胞内O-GlcNAc水平的有用工具，从而为深入了解O-GlcNAc修饰蛋白的生理作用提供有价值的见解。
