An Hyun Joo, Peavy Thomas R, Hedrick Jerry L, Lebrilla Carlito B
Department of Chemistry and Section of Molecular and Cell Biology, University of California, Davis, California 95616, USA.
Anal Chem. 2003 Oct 15;75(20):5628-37. doi: 10.1021/ac034414x.
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.
本文描述了一种基于非特异性蛋白酶解、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）相结合的方法，用于表征糖蛋白中的糖基化位点和寡糖异质性。糖蛋白用链霉蛋白酶消化，主要产生糖肽和氨基酸。非糖基化的肽片段易被链霉蛋白酶完全消化成其组成氨基酸。空间位阻阻止了与聚糖相连的肽部分的消化。糖肽通过固相萃取进行脱盐和浓缩，并通过MALDI MS分析。使用PNGase F从糖蛋白中释放聚糖后，也通过MALDI MS分析寡糖。通过从糖肽的质量中减去PNGase F处理产生的聚糖的质量来鉴定糖肽的肽部分。使用具有已知寡糖结构的糖蛋白、核糖核酸酶B和鸡卵清蛋白验证了该实验策略。然后使用该程序来确定糖基化模式未知的糖蛋白，即非洲爪蟾卵皮质颗粒凝集素的N-糖基化位点和位点异质性。该程序可用于确定糖蛋白的蛋白质位点异质性和寡糖部分的结构异质性。
