Manri Naomi, Takegawa Yasuhiro, Fujitani Naoki, Kaneko Akihito, Hirabayashi Atsumu, Nishimura Shin-Ichiro, Sakamoto Takeshi
Central Research Laboratory, Hitachi, Ltd, Kokubunji, Tokyo, Japan.
Anal Sci. 2012;28(7):723-7. doi: 10.2116/analsci.28.723.
A mass-spectrometric method for a de novo determination of O-glycosylation heterogeneity was developed. We used a mild fragmentation technique, electron capture dissociation (ECD), which enables the determination of glycosylation sites as well as peptide sequencing. To demonstrate the correct identification of glycopeptides, we prepared a series of glycopeptides with the same peptide sequence and 6 different glycan modifications. ECD spectra were obtained at various electron energies, and were analyzed with the Mascot database-search engine. The obtained candidate glycopeptides were further validated by confirming the spectral overlap of ECD fragment peaks with the theoretical peaks. The results indicate that all glycopeptides were unambiguously identified, including glycosylation sites by combining ECD results with different electron energies for each glycopeptide.
开发了一种用于从头测定O-糖基化异质性的质谱方法。我们使用了一种温和的碎片化技术，即电子捕获解离（ECD），它能够确定糖基化位点以及进行肽测序。为了证明对糖肽的正确鉴定，我们制备了一系列具有相同肽序列和6种不同聚糖修饰的糖肽。在不同电子能量下获得ECD光谱，并使用Mascot数据库搜索引擎进行分析。通过确认ECD片段峰与理论峰的光谱重叠，对获得的候选糖肽进行进一步验证。结果表明，通过将ECD结果与每种糖肽的不同电子能量相结合，所有糖肽都能被明确鉴定，包括糖基化位点。
