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) 质谱法，结合蛋白水解消化 和 BPLC 分离，已用于验证 cDNA 预测的氨基 鸡II型胶原蛋白的酸序列。 II型胶原蛋白 分子由三个相同的多肽链组成[α， (11) 链] 交织成三螺旋。 单三螺旋 该结构域包含约 96% 的氨基酸序列，并由以下组成 甘氨酸-X-Y (G-X-Y) 形式的重复三肽。 螺旋状 结构域的 N 端和 C 端两侧均带有短 称为端肽的非螺旋结构域。 分子量为 未修饰的鸡 11 型胶原蛋白三螺旋预测 cDNA序列为294 kDa。 发生翻译后修饰 在许多地点；研究最好的是脯氨酸的羟基化和 G-X-Y序列的Y位置的赖氨酸，糖基化 羟基化赖氨酸和赖氨酸之间的螺旋间交联 和羟基赖氨酸。 该蛋白质的质谱分析是 非常具有挑战性，因为有几个因素会产生异质性。 大量潜在位点的不完全修饰 翻译后修饰产生多种形式。 期间 胃蛋白酶溶解鸡胸骨11型胶原蛋白 消化时，C 端端肽可能在几个位置被切割 不同的点，产生额外的异质性。 消化 也可以从交联肽生成“标签” 附着在主链上。 鸡11型胶原蛋白已火热 变性并用内切蛋白酶 Lys-C 消化。 Lys-C 肽 通过反相 FIPLC 分离、收集并通过 MALDI-TOF MS 和 ESI MS。 共有131种不同的肽 分子量范围为 711 Da 至 14,027 Da 迄今为止已确定。 MS/MS 分析和二次消化已 确定肽序列并定位修饰 肽内的位点。 我们的质谱分析有 鉴定出许多羟基化和糖基化位点，并已 鉴定出几个C-末端。 这一分析使我们能够确认 从 cDNA 序列预测的大部分残基并校正 很少有错误分配结果表明，在 羟基化和糖基化以及随之而来的转变 色谱行为。