Laboratory of Glycobiology, The Noguchi Institute, 1-8-1 Kaga, Itabashi, Tokyo, 173-0003, Japan.
Anal Chem. 2010 Oct 15;82(20):8738-43. doi: 10.1021/ac101555a.
Glycoproteomics holds the promise of new advances in medical technology. However, mass spectrometry has limitations for the structural determination of glycosylated peptides because the hydrophilic nature of the oligosaccharide moiety in glycopeptides is disadvantageous for ionization, and glycopeptides ionize much less readily than nonglycosylated peptides. Therefore, conventional proteomics tools cannot detect altered glycosylation on proteins. Here, we describe an on-plate pyrene derivatization method using 1-pyrenyldiazomethane for highly sensitive matrix-assisted laser/desorption ionization-tandem mass spectrometry (MALDI-MS(n)) of glycopeptides in amounts of less than 100 fmol. This derivatization is unique, as the pyrene groups are easily released from glycopeptides during ionization when 2,5-dihydroxybenzoic acid is used as a matrix. As a result, most ions are observed as the underivatized form on the spectra. At the same time, pyrene derivatization dramatically reduces the ionization of peptides. Thus, for glycopeptides in a mixture of abundant peptides, we could obtain MS spectra in which the signals of glycopeptides were intense enough for subjection to MS(n) in order to determine the structures of both glycan and peptide. Finally, we show that the glycopeptides derived from as little as 1 ng of prostate specific antigen can be detected by this method.
糖蛋白质组学有望推动医学技术的新进展。然而，由于糖肽中寡糖部分的亲水性不利于离子化，而且糖肽的离子化程度远低于非糖肽，因此质谱技术在糖肽结构测定方面存在局限性。因此，传统的蛋白质组学工具无法检测蛋白质上发生的糖基化改变。在这里，我们描述了一种在板上的芘衍生化方法，使用 1-芘基重氮甲烷对少于 100 飞摩尔的糖肽进行高灵敏度基质辅助激光解吸/串联质谱（MALDI-MS(n)）分析。这种衍生化方法很独特，因为当使用 2,5-二羟基苯甲酸作为基质时，芘基很容易在糖肽离子化过程中释放出来。因此，在光谱上观察到的大多数离子都是未衍生的形式。同时，芘衍生化极大地降低了肽的离子化程度。因此，对于富含肽的糖肽混合物，我们可以获得糖肽信号足够强的 MS 谱，以便进行 MS(n)以确定聚糖和肽的结构。最后，我们证明了通过这种方法可以检测到少至 1ng 的前列腺特异性抗原衍生的糖肽。
