Lattová Erika, Chen Vincent C, Varma Sonal, Bezabeh Tedros, Perreault Hélène
Chemistry Department, University of Manitoba, Winnipeg, MB, Canada R3T 2N2.
Rapid Commun Mass Spectrom. 2007;21(10):1644-50. doi: 10.1002/rcm.3007.
The significance of glycoproteins in living systems instigates the ceaseless expansion of new techniques and procedures for the analysis of biological samples. Many of these applications are focused on improving the detection limit of analyzed material. In a previous study, we described a procedure for the detection of oligosaccharides cleaved from tryptic glycopeptides. Treatment of deglycosylated fractions with phenylhydrazine gave rise to peaks consistent with labeled glycans, and both types of compounds--deglycosylated peptides and oligosaccharides--were recorded from one spot and observed in one matrix-assisted laser desorption/ionization (MALDI) mass spectrum for the first time. Here, we added an additional step to this simple procedure of deglycosylating glycopeptides directly from the target spot of the first analyzed glycosylated peptides. For the purpose of this new study, a mixture of 2-aza-2-thiothymine and phenylhydrazine hydrochloride showed to be an excellent matrix for glycopeptides, oligosaccharides, deglycosylated peptides and moreover it allowed PNGaseF to be active enough to cleave oligosaccharides from peptides. The efficiency of this procedure is demonstrated on a series of intact glycoproteins and on the analysis of tryptic peptides obtained from IgG and total mouse serum. This one-step on-target deglycosylation method with subsequent derivatization on the same spot makes MALDI-MS analyses of glycopeptides fast, simple and accessible for biological samples, where classical procedures cannot produce useful results.
糖蛋白在生命系统中的重要性促使用于生物样品分析的新技术和程序不断扩展。这些应用中的许多都集中在提高被分析物质的检测限上。在先前的一项研究中，我们描述了一种检测从胰蛋白酶糖肽上裂解下来的寡糖的方法。用苯肼处理去糖基化部分会产生与标记聚糖一致的峰，并且首次在一个基质辅助激光解吸/电离（MALDI）质谱中从一个斑点记录到两种类型的化合物——去糖基化肽和寡糖。在这里，我们在这个直接从第一个分析的糖基化肽的目标斑点对糖肽进行去糖基化的简单程序中增加了一个额外步骤。为了这项新研究的目的，2-氮杂-2-硫代胸腺嘧啶和盐酸苯肼的混合物被证明是用于糖肽、寡糖、去糖基化肽的优秀基质，而且它能使N-糖苷酶F具有足够的活性从肽上裂解寡糖。该程序的效率在一系列完整糖蛋白以及从IgG和小鼠全血清获得的胰蛋白酶肽的分析中得到了证明。这种在同一斑点上进行一步原位去糖基化并随后衍生化的方法，使得对糖肽的MALDI-MS分析对于生物样品来说快速、简单且可行，而传统程序无法产生有用的结果。
