MAPPING PEPTIDE BINDING SITES OF SP & SK RECEPTORS

SP 肽结合位点图谱

• 批准号: 6345209
• 负责人: NORMAN D BOYD
• 金额: $ 0.62万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6345209
• 关键词: Chordata; arthritis; biological products; biomedical resource; connective tissue; proteins; skeletal system; spectrometry; structural biology; technology /technique

Electrospray (ESI) and matrix-assisted laser desorption/ionization (NLALDI) mass spectrometry, in combination with proteolyic digestion and BPLC separation, have been used to verify the cDNA-predicted amino acid sequence of chicken type II collagen. The type II collagen molecule is composed of three identical polypeptide chains [alpha, (11) chains] intertwined in a triple helix. The single triple helical domain comprises about 96% of the amino acid sequence and consists of repeating tripeptides of the form glycine-X-Y (G-X-Y). The helical domain is flanked on both the N-terminal and C-terminal ends by short non-helical domains known as telopeptides. Themolecular weight for the unmodified chicken type 11 collagen triple helix predicted from the cDNA sequence is 294 kDa. Posttranslational modifications occur at numerous sites; the best studied are hydroxylation of prolines and lysines in the Y position of the G-X-Y sequence, glycosylation of hydroxylated lysines, and interhelical cross-linkages between lysines and hydroxylysines. Mass spectrometric analysis of this protein is very challenging because several factors produce heterogeneity. Incomplete modification at the large number of potential sites for post-translational modifications produces a variety of forms. During the solubilization of type 11 collagen from chicken sterna by pepsin digestion, the C-terminal telopeptide may be cleaved at several different points, producing additicrnal heterogeneity. The digestion may also generate "tags" from the crosslinked peptides that are attached to the main chain. Chicken type 11 collagen hasbeen heat denatured and digested with endoproteinase Lys-C. The Lys-C peptides were separated by reversed-phase FIPLC, collected and examined by MALDI-TOF MS and ESI MS. A total of 131 different peptides with molecular weights ranging from 711 Da to 14,027 Da have been identified to date. MS/MS analyses and secondary digestions have been performed to determine peptide sequence and to locate modification sites within a peptide. Our mass spectrometric analysis has identified numerous sites of hydroxylation and glycosylation and has identified several C-termini. This analysis has enabled us to confirm most of the residues predicted from the cDNA sequence and to correct a few misassignments The results show a high degree of heterogeneity in hydroxylation and glycosylation with attendant shifts in chromatographic behavior.

电喷雾（ESI）和基质辅助激光解吸/电离 （Nlaldi）质谱，结合蛋白质消化 和BPLC分离已被用来验证cDNA预测的氨基 鸡型II胶原蛋白的酸序列。 II型胶原蛋白 分子由三个相同的多肽链组成[alpha， （11）链条在三重螺旋中交织在一起。 单三重螺旋 域约占氨基酸序列的96％，由 甘氨酸-X-Y（G-X-Y）形式的重复三肽。 螺旋 域在N末端和C末端都处于侧面 非螺旋结构域称为端肽。 分子重量 未修饰的鸡肉类型11胶原蛋白三重螺旋可预测 cDNA序列为294 kDa。 翻译后修饰发生 在许多地点；研究最好的是脯氨酸的羟基化和 G-X-Y序列Y位置的赖氨酸，糖基化的糖基化 羟化赖氨酸和赖氨酸之间的旋转交联 和羟基。 该蛋白的质谱分析是 非常具有挑战性，因为几个因素会产生异质性。 大量潜在地点的修改不完全 翻译后修饰会产生多种形式。 期间 胃蛋白酶从鸡斯特尔纳溶解11型胶原蛋白 消化，C末端端肽可以在几个 不同的点，产生添加的异质性。 消化 也可能从交联的肽中生成“标签” 附在主链上。 鸡肉类型11胶原蛋白Hosbeen Heat 用内蛋白酶Lys-C进行变性和消化。 Lys-C肽 通过反相FIPLC分离，收集并检查 MALDI-TOF MS和ESI MS。 共有131种不同的肽 从711 DA到14,027 DA的分子量已经 迄今已确定。 MS/MS分析和次要消化已经 执行以确定肽序列并定位修饰 肽内的位置。 我们的质谱分析具有 鉴定出许多羟基化和糖基化位点，并具有 确定了几个C末端。 这种分析使我们得以确认 从cDNA序列预测的大多数残基并纠正A 很少的错误纠正结果显示出高度的异质性 随随之而来的羟基化和糖基化 色谱行为。