MAPPING PEPTIDE BINDING SITES OF SP & SK RECEPTORS

电喷雾和基质辅助激光解吸/电离 (NLALDI)质谱学，结合蛋白质分解消化 和BPLC分离，用来验证所预测的氨基酸 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]交织成三股螺旋。单一的三重螺旋 结构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。螺旋线 结构域位于N-末端和C-末端的两侧，简称 称为端肽的非螺旋结构域。分子量与分子质量 未修饰的鸡11型胶原蛋白三螺旋结构预测 该基因的全长为294 kDa。翻译后修饰发生 在许多位置；最好的研究是脯氨酸的羟基化和 G-X-Y序列中Y位的赖氨酸，糖基化 羟基化赖氨酸和赖氨酸之间的螺旋间交联键 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。 对大量潜在站点的修改不完整 翻译后修饰语产生了多种形式。在.期间 胃酶对鸡胸腺11型胶原的增溶作用 消化时，C-末端的端肽可能会被几个 不同的点，产生额外的异质性。消化 也可以从具有以下特性的交联肽中产生“标签” 固定在主链上。鸡肉11型胶原蛋白已过热 用内切酶Lys-C变性和消化。Lys-C多肽 用反相FIPLC分离，收集和检测 MALDI-TOF MS和ESI MS共131种不同的多肽 分子量从711 Da到14,027 Da不等 到目前为止已经确定了。MS/MS分析和二次消化已被 用于确定多肽序列并定位修饰 多肽中的位置。我们的质谱分析已经 确定了大量的羟化和糖基化位点，并已 确定了几个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和纠正a 很少的错误分配结果显示出高度的异质性 羟基化和糖基化及其伴随的变化 层析行为。

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.