Weitzhandler M, Kadlecek D, Avdalovic N, Forte J G, Chow D, Townsend R R
Dionex Corporation, Sunnyvale, California 94088.
J Biol Chem. 1993 Mar 5;268(7):5121-30.
We have developed an intermediate method toward the complete carbohydrate analysis of proteins, which should be universally applicable to all proteins and independent of sample matrix. Using only Coomassie Blue-stained proteins which have been electroblotted onto polyvinylidene fluoride membranes, we report a strategy for: (i) determining unequivocally whether a protein is glycosylated; (ii) obtaining a complete monosaccharide composition; (iii) oligosaccharide mapping which separates most forms according to size, charge and isomerity; and (iv) sequentially releasing and analyzing specific classes of oligosaccharides with endoglycosidases. The method was shown to be applicable to a variety of well characterized soluble glycoproteins and to the membrane-bound protein, the gastric H+, K(+)-ATPase. The monosaccharide composition of the H+,K(+)-ATPase revealed the absence of N-acetylneuraminic or N-glycolylneuraminic acids and a monosaccharide composition which indicated O-linked sugar chains. Oligomannosidic/hybrid and biantennary oligosaccharides were sequentially released and analyzed from one electroblotted band of recombinant tissue plasminogen activator using endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F2, respectively. Sialylated polylactosamine structures were identified and quantified by analyzing high performance liquid chromatography profiles of oligosaccharides first released by peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase and then treated with endo-beta-galactosidase, using a single, stained band of recombinant erythropoietin. This recombinant erythropoietin was found to contain eight times more tetrasialylated oligosaccharides than previously reported (Sasaki, H., Bothner, B., Dell, A., and Fukuda, M. (1987) J. Biol. Chem. 262, 12059-12076); 47% of released oligosaccharides were identified as polylactosamine structures.
我们已经开发出一种用于蛋白质完整碳水化合物分析的中间方法，该方法应普遍适用于所有蛋白质且与样品基质无关。仅使用已电转印到聚偏二氟乙烯膜上的考马斯亮蓝染色蛋白质，我们报告了一种策略，用于：（i）明确确定蛋白质是否糖基化；（ii）获得完整的单糖组成；（iii）寡糖图谱分析，根据大小、电荷和异构性分离大多数形式的寡糖；以及（iv）用内切糖苷酶顺序释放和分析特定类别的寡糖。该方法已证明适用于多种特征明确的可溶性糖蛋白以及膜结合蛋白——胃H⁺，K⁺-ATP酶。H⁺，K⁺-ATP酶的单糖组成显示不存在N-乙酰神经氨酸或N-糖基神经氨酸，且单糖组成表明存在O-连接糖链。分别使用内切β-N-乙酰氨基葡糖苷酶H和内切β-N-乙酰氨基葡糖苷酶F2，从重组组织型纤溶酶原激活剂的一条电转印条带中顺序释放并分析了低聚甘露糖型/杂合型和二天线型寡糖。通过分析寡糖的高效液相色谱图谱来鉴定和定量唾液酸化的多乳糖胺结构，这些寡糖首先由肽-N4-（N-乙酰-β-D-葡糖胺基）天冬酰胺酶释放，然后用内切β-半乳糖苷酶处理，使用重组促红细胞生成素的一条染色条带。发现这种重组促红细胞生成素含有的四唾液酸化寡糖比先前报道的多八倍（佐佐木，H.，博特纳，B.，戴尔，A.，和福田，M.（1987年）《生物化学杂志》262，12059 - 12076）；47%的释放寡糖被鉴定为多乳糖胺结构。
