STRUCT OF POST TRANSLATIONAL MODIFICATIONS OF CF TRANSMEMBRANE CONDUCTANCE

主要翻译后修饰的结构解析 重组CFTR（rCFTR）。 多肽定位 将测定rCFTR的磷酸化和糖基化位点 使用灵敏的质谱分析法 CFTR蛋白和 将使用来自核心I的蛋白质片段。 我们的初步 对另一种膜糖蛋白CHIP-28的研究表明， 证明我们的方法可以用于pmol的分析 CFTR的数量。 完整的磷酸盐基团的数量 rR结构域在E.大肠杆菌，将使用 基质辅助激光解吸，飞行时间质谱。 磷酸化肽，来自RR结构域的蛋白水解酶， 将被识别和表征（例如，磷酸根数 使用微孔HPLC/电喷雾电离质谱 光谱法 磷酸化丝氨酸残基的位置 将通过使用串联测序磷酸化肽来确定 用高能碰撞诱导解离进行质谱分析。 一 类似的方法将用于定位磷酸化残基 哺乳动物和昆虫细胞的rCFTR。 多肽定位 将从精确质量推导出N-和0糖基化位点的 测定（上午1点以内）蛋白水解产生的糖肽 在昆虫（Sf 9）和哺乳动物细胞中表达的rCFTR的启动子。 的 在昆虫细胞中表达的来自rCFTR的寡糖将被 在它们释放后使用自动肼解进行测序。 这些 研究将确定昆虫细胞糖基化的程度 可作为哺乳动物细胞糖基化CFTR的模型。 哺乳动物CFTR寡糖的结构特征 将使用“低聚糖”组合来确定来源 作图”和外切糖苷酶分析。 CFTR的调查 磷酸化，迄今为止已经产生了模糊的发现， 对于了解其作为氯离子通道的功能及其 通过蛋白激酶和磷酸酶调节。 更详细的研究 的CFTR碳水化合物是必要的，以更好地了解 各种突变体的细胞内运输异常， CF患者

Structural elucidation of major post-translational modifications of recombinant CFTR (rCFTR) is proposed. Polypeptide localization of phosphorylation and glycosylation sites of rCFTR will be determined using a sensitive mass spectrometric-based strategy. CFTR proteinand protein fragments from Core I will be utilized. Our preliminary studies with another membrane glycopro tein, CHIP-28, have demonstrated that our methodology can be used for the analysis of pmol quantities of CFTR. The number of phos phate groups on intact rR-domain, expressed in E. coli, will be determined using matrix-assisted laser desorption, time of flight mass spectrometry. Phosphorylated peptides, from proteolytic digests of the rR-domain, will be identified and characterized (e.g., number of phosphate groups) using micro-bore HPLC/electro spray ionization mass spectrometry. The location of the phos phorylated serine residues will be determined by sequencing phos phorylated peptides using tandem mass spectrometry with high energy collision induced dissocation. A similar approach will be used to localize the phosphorylated residues of rCFTR from mammal ian and insect cells. The polypeptide location of the N- and 0 glycosylation sites will be deduced from accurate mass determina tion (within 1 a.m.u.) of glycopeptides from proteolytic digests of rCFTR, expressed in insect (Sf9) and mammalian cells. The oligosaccharides from rCFTR, expressed in insect cells, will be sequenced after their release using automated hydrazinolysis. These studies will ascertain the extent to which insect cell glycosylation can be used as a model for mammalian cell glycosyla tion of CFTR. Structural features of the oligosaccharides of CFTR from mammalian sources will be determined using a combination of "oligosaccharide mapping" and exoglycosidase analysis. Investiga tions of CFTR phosphorylation, which heretofore has yielded ambiguous findings, is essential for understanding its function as a chloride channel and its regulation by protein kinases and phosphatases. More detailed studies of CFTR carbohydrate are necessary to understand better the abnormalities of intracellular trafficking of the various mutants in CF patients.