Wada Yoshinao
Department of Molecular Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health.
Mass Spectrom (Tokyo). 2012;1(2):A0008. doi: 10.5702/massspectrometry.A0008. Epub 2012 Nov 2.
Mucin-type O-glycosylation is a major posttranslational modification of proteins. The level of O-glycosylation at a site could be useful in terms of evaluating various disease conditions. To address the feasibility of measuring O-glycosylation levels based on the glycopeptide ion intensity in a mass spectrum, apolipoprotein CIII (apoC3), a protein that contains a single core-1 O-glycan Gal-GalNAc disaccharide was analyzed by matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). The intensity of protonated ions for an equimolar mixture of desialylated and deglycosylated apoC3s were the same in linear TOF measurements. No substantial in-source decay, including the cleavage of the protein-sugar linkage was observed. The glycopeptide derived from apoC3 and the unglycosylated counterpart, when analyzed by MALDI reflectron TOF MS indicated that post-source decay was minimal. These collective findings demonstrate the feasibility of label-free quantitation of O-glycan occupancy by MS when the glycans are small and neutral. This method provides a tool for use in glycoproteomics as a complement of our previous report (DOI: 10.1021/pr900913k) for calculating the saccharide composition of O-glycans.
粘蛋白型O-糖基化是蛋白质主要的翻译后修饰。某一位点的O-糖基化水平对于评估各种疾病状况可能具有参考价值。为了探讨基于质谱中糖肽离子强度测定O-糖基化水平的可行性，我们采用基质辅助激光解吸电离（MALDI）飞行时间（TOF）质谱（MS）分析了载脂蛋白CIII（apoC3），该蛋白含有一个核心-1 O-聚糖Gal-GalNAc二糖。在直线型TOF测量中，去唾液酸化和去糖基化apoC3等摩尔混合物的质子化离子强度相同。未观察到明显的源内衰变，包括蛋白质-糖键的断裂。当通过MALDI反射式TOF MS分析时，源自apoC3的糖肽和未糖基化对应物表明源后衰变最小。这些共同发现证明，当聚糖小且呈中性时，通过质谱对O-聚糖占据情况进行无标记定量是可行的。该方法为糖蛋白质组学提供了一种工具，可作为我们之前报告（DOI: 10.1021/pr900913k）中计算O-聚糖糖组成的补充。
