Determination of N-glycosylation sites and site heterogeneity in glycoproteins
Determination of N-glycosylation sites and site heterogeneity in glycoproteins
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DOI：
10.1021/ac034414x
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发表时间：
2003-10-15
影响因子：
7.4
通讯作者：
Lebrilla, CB
中科院分区：
文献类型：
--
作者：
An, HJ;Peavy, TR;Lebrilla, CB
描述了一种基于非特异性蛋白水解、去糖基化和基质辅助激光解吸/电离傅里叶变换质谱（MALDI-FT MS）相结合的糖蛋白中糖基化位点和寡糖异质性的表征方法。用链霉蛋白酶消化糖蛋白，主要产生糖肽和氨基酸。非糖基化肽片段易于被链霉蛋白酶完全消化成其组成氨基酸。空间位阻抑制了与聚糖连接的肽部分的消化。使用固相萃取对糖肽进行脱盐和浓缩，并通过MALDI MS进行分析。使用PNGase F从糖蛋白中释放聚糖后，还通过MALDI MS对寡糖进行分析。通过从糖肽质量中减去PNGase F处理产生的聚糖质量，鉴别糖肽的肽部分。使用已知寡糖结构的糖蛋白、核糖核酸酶B和鸡卵清蛋白验证实验策略。然后，这个程序被用来确定的N-糖基化位点和糖基化模式是未知的，即，非洲爪蟾卵皮质颗粒凝集素的糖蛋白的网站异质性。该程序可用于确定糖蛋白的寡糖部分的蛋白质位点异质性和结构异质性。
An approach for the characterization of glycosylation sites and oligosaccharide heterogeneity in glycoproteins based on a combination of nonspecific proteolysis, deglycosylation, and matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FT MS) is described. Glycoproteins were digested with Pronase yielding primarily glycopeptides and amino acids. Nonglycosylated peptide fragments were susceptible to complete Pronase digestion to their constituent amino acids. Steric hindrance prohibited the digestion of the peptide moiety attached to the glycan. Glycopeptides were desalted and concentrated using solid-phase extraction and analyzed by MALDI MS. The oligosaccharides were also analyzed by MALDI MS after releasing the glycans from glycoproteins using PNGase F. The peptide moiety of the glycopeptides was identified by subtracting the masses of the glycans derived from PNGase F treatment from the masses of the glycopeptides. The experimental strategy was validated using glycoproteins with known oligosaccharide structures, ribonuclease B and chicken ovalbumin. This procedure was then used to determine the N-glycosylation sites and site heterogeneity of a glycoprotein whose glycosylation pattern was unknown, namely, the Xenopus laevis egg cortical granule lectin. This procedure is useful for determining protein site heterogeneity and structural heterogeneities of the oligosaccharide moiety of glycoproteins.