MAPPING PEPTIDE BINDING SITES OF SP & SK RECEPTORS

SP 肽结合位点图谱

• 批准号: 6206404
• 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: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6206404
• 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，结合蛋白水解消化 和BPLC分离，已被用来验证cDNA预测的氨基酸 鸡II型胶原蛋白的酸性序列。 II型胶原蛋白 分子由三条相同的多肽链组成[α， (11)链]在三重螺旋中缠绕。 单三螺旋 结构域包含约96%的氨基酸序列，并且由以下组成： 甘氨酸-X-Y（G-X-Y）形式的重复三肽。 螺旋 结构域在N-末端和C-末端上都侧接短的 称为端肽的非螺旋结构域。 分子量 未修饰的鸡11型胶原蛋白三螺旋预测从 cDNA序列为294 kDa。 翻译后修饰发生在 在许多位点;研究得最好的是脯氨酸的羟基化， G-X-Y序列的Y位置上的赖氨酸， 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。 在大量的潜在地点进行不完全的修改， 翻译后修饰产生多种形式。 期间 胃蛋白酶对鸡胸骨11型胶原蛋白增溶作用 消化后，C-末端端肽可以在几个位点被切割， 不同的点，产生额外的异质性。 消化 也可以从交联肽产生“标签”， 附在主链上。 鸡11型胶原蛋白已被加热 变性并用内切蛋白酶Lys-C消化。 Lys-C肽 通过反相HPLC分离，收集并通过HPLC检测。 MALDI-TOF MS和ESI MS。 分子量范围为711 Da至14，027 Da， 识别日期。 MS/MS分析和二级数字化已被 进行测定肽序列和定位修饰 肽内的位点。 我们的质谱分析 鉴定了许多羟基化和糖基化位点， 鉴定了几个C-末端。 通过分析我们可以确认 从cDNA序列预测的大多数残基， 结果显示，在不同的实验条件下， 羟基化和糖基化，伴随着 色谱行为