Irungu Janet, Go Eden P, Zhang Ying, Dalpathado Dilusha S, Liao Hua-Xin, Haynes Barton F, Desaire Heather
Department of Chemistry, University of Kansas, 1251 Wescoe Hall Drive, Lawrence, KS 66045, USA.
J Am Soc Mass Spectrom. 2008 Aug;19(8):1209-20. doi: 10.1016/j.jasms.2008.05.010. Epub 2008 May 24.
Defining the structures and locations of the glycans attached on secreted proteins and virus envelope proteins is important in understanding how glycosylation affects their biological properties. Glycopeptide mass spectrometry (MS)-based analysis is a very powerful, emerging approach to characterize glycoproteins, in which glycosylation sites and the corresponding glycan structures are elucidated in a single MS experiment. However, to date there is not a consensus regarding which mass spectrometric platform provides the best glycosylation coverage information. Herein, we employ two of the most widely used MS approaches, online high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC/ESI-MS) and offline HPLC followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), to determine which of the two approaches provides the best glycosylation coverage information of a complex glycoprotein, the group M consensus HIV-1 envelope, CON-S gp140DeltaCFI, which has 31 potential glycosylation sites. Our results highlight differences in the informational content obtained between the two methods such as the overall number of glycosylation sites detected, the numbers of N-linked glycans present at each site, and the type of confirmatory information obtained about the glycopeptide using MS/MS experiments. The two approaches are quite complementary, both in their coverage of glycopeptides and in the information they provide in MS/MS experiments. The information in this study contributes to the field of mass spectrometry by demonstrating the strengths and limitations of two widely used MS platforms in glycoprotein analysis.
确定分泌蛋白和病毒包膜蛋白上连接的聚糖的结构和位置，对于理解糖基化如何影响它们的生物学特性至关重要。基于糖肽质谱（MS）的分析是一种非常强大且新兴的糖蛋白表征方法，在一次质谱实验中即可阐明糖基化位点及相应的聚糖结构。然而，迄今为止，关于哪种质谱平台能提供最佳的糖基化覆盖信息尚无共识。在此，我们采用两种最广泛使用的质谱方法，即在线高效液相色谱 - 电喷雾电离质谱（HPLC/ESI-MS）和离线HPLC后接基质辅助激光解吸/电离质谱（MALDI-MS），来确定这两种方法中哪种能为具有31个潜在糖基化位点的复杂糖蛋白——M组HIV-1包膜蛋白共识序列CON-S gp140DeltaCFI提供最佳的糖基化覆盖信息。我们的结果突出了两种方法所获得信息内容的差异，例如检测到的糖基化位点总数、每个位点存在的N-连接聚糖数量，以及通过串联质谱（MS/MS）实验获得的关于糖肽的确认信息类型。这两种方法在糖肽覆盖范围和MS/MS实验所提供的信息方面都具有很强的互补性。本研究中的信息通过展示两种广泛使用的质谱平台在糖蛋白分析中的优势和局限性，为质谱领域做出了贡献。
