Rademaker G J, Pergantis S A, Blok-Tip L, Langridge J I, Kleen A, Thomas-Oates J E
Department of Mass Spectrometry, Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands.
Anal Biochem. 1998 Mar 15;257(2):149-60. doi: 10.1006/abio.1997.2548.
A sensitive protocol for unambiguously and positively identifying O-glycosylation sites in glycopeptides is described, based on beta-elimination of the glycan chain(s) using NH4OH. On glycan elimination, NH3 is incorporated into the amino acid residue(s) to which the glycan(s) had been attached, to yield a modified amino acid residue having a distinct mass. Electrospray ionization collision-induced dissociation tandem mass spectrometry allows the released, modified peptide to be sequenced and the site(s) of the modified amino acid residue(s) to be identified. The protocol has been optimized using a series of structurally related O-glycopeptides, and standard conditions are recommended for handling unknowns. We demonstrate that site determination can be achieved using as little as 1 pmol of starting material.
描述了一种用于明确且肯定地鉴定糖肽中O-糖基化位点的灵敏方法，该方法基于使用NH₄OH对聚糖链进行β-消除。在聚糖消除过程中，NH₃被掺入到曾连接有聚糖的氨基酸残基中，从而产生具有独特质量的修饰氨基酸残基。电喷雾电离碰撞诱导解离串联质谱法可对释放出的修饰肽进行测序，并鉴定修饰氨基酸残基的位点。该方法已通过一系列结构相关的O-糖肽进行了优化，并推荐了用于处理未知样品的标准条件。我们证明，使用低至1皮摩尔的起始材料即可实现位点测定。
