Process and Product Development, Amgen, Seattle, WA 98119, USA.
Anal Biochem. 2010 May 1;400(1):25-32. doi: 10.1016/j.ab.2010.01.026. Epub 2010 Jan 25.
The site-specific characterization of the complex glycans in multiglycosylated proteins requires developing methods where the carbohydrates remain covalently bound to the protein. The complexity in the carbohydrate composition of alpha(1)-acid glycoprotein (AAG) makes it an ideal model protein for such development. AAG has five N-asparaginyl-linked glycosylation sites, each varying in its bi-, tri-, and tetraantennary glycan content. We present an on-line liquid chromatography/mass spectrometry (LC/MS) method that uses high-low cone voltage switching for in-source fragmentation to determine the structures of the complex glycans present on each site for the two gene products of AAG. High cone voltage caused carbohydrate fragmentation, leading to the generation of signature carbohydrate ions that we used as markers to identify the glycopeptides. Low cone voltage produced minimal carbohydrate fragmentation and enabled the identification and quantification of the intact oligosaccharide structures on each glycopeptide based on its monoisotopic mass and intensity. Quantitation was accomplished by using the intensities of peaks from deconvoluted and deisotoped mass spectra or from the areas of the extracted ion chromatograms from the tryptic peptide maps. The combined results from the two methods can be used to better characterize and quantitate site heterogeneity in multiglycosylated proteins.
多聚糖蛋白中特定位置的复杂聚糖的特征化需要开发出一些方法，使碳水化合物仍然与蛋白质共价结合。α(1)-酸性糖蛋白 (AAG) 的碳水化合物组成的复杂性使其成为此类开发的理想模型蛋白。AAG 有五个 N-天冬酰胺连接的糖基化位点，每个位点的二、三、四天线聚糖含量都不同。我们提出了一种在线液相色谱/质谱 (LC/MS) 方法，该方法使用高低锥电压切换进行源内碎裂，以确定 AAG 两种基因产物中每个位点存在的复杂聚糖的结构。高锥电压导致碳水化合物碎裂，产生特征性的碳水化合物离子，我们将其用作标记物来识别糖肽。低锥电压几乎不会引起碳水化合物碎裂，从而能够根据每个糖肽的单同位素质量和强度来识别完整的寡糖结构，并对其进行定量。定量是通过使用解卷积和去同位素化质谱的峰强度或从胰蛋白酶肽图谱的提取离子色谱图的面积来完成的。两种方法的综合结果可用于更好地表征和定量多聚糖蛋白中的位置异质性。
