鹿皮胶Ⅰ型胶原赖氨酸位点的羟基化与O-糖基化修饰分析

Analysis of hydroxylation and O-glycosylation on lysine sites in deer-hide gelatin

该文采用纳升液相-串联质谱法对鹿皮胶样品进行分析,以PEAKS Studio蛋白质搜库鉴定软件对鹿皮胶赖氨酸位点的羟基化与O-糖基化修饰进行全面鉴定,明确鹿皮胶Ⅰ型胶原蛋白α1链(COL1A1)与α2链(COL1A2)发生羟基化与O-糖基化的主要位点、类型及数量。结果从4批次鹿皮胶样品中共鉴定了5284个肽段信息,主要来源于COL1A1与COL1A2,其中羟基化赖氨酸(hydroxylysine,Hyl)共449个,半乳糖基化赖氨酸(galactosyl-hydroxylysine,Gal-Hyl)共442个,葡萄糖半乳糖基化赖氨酸(glucosyl-galactosyl-hydroxylysine,Glc-Gal-Hyl)共449个。鹿皮胶的Ⅰ型胶原发生羟基化与O-糖基化的主要位点有:N-末端肽的α1-9N与α2-5N,三螺旋结构域的α1-87,α1-174,α1-930,α2-87,α2-174,α2-933,C-末端肽的α1-16C。这些羟基化与糖基化赖氨酸与Ⅰ型胶原的三螺旋结构及胶原纤维形成密切相关,而鹿皮经高温高压熬制的过程破坏胶原的共价连接有利于胶原蛋白与胶原肽的溶出,形成具有糖基化修饰的胶原肽类成分。该文研究为鹿皮胶的功效物质基础的深入揭示提供了研究思路与方法依据,具有较好的理论意义与应用价值。

Nano-LC MS/MS was used to analyze trypsin digested deer-hide gelatin(DHG)samples,hydroxylation and O-glycosylation on lysine sites of DHG were comprehensive identified by using PEAKS Studio software.The sites,sorts and amounts of hydroxylation and O-glycosylation on TypeⅠcollagenα1 chain(COL1 A1)andα2 chain(COL1 A2)of DHG were revealed.As a result,5284 peptides were identified from DHG samples,which were mainly from COL1 A1 and COL1 A2.Among these peptides,there were 449 peptides with hydroxylysine,442 with galactosyl-hydroxylysine,449 with glucosyl-galactosyl-hydroxylysine.The major modified sites of hydroxylation and O-glycosylation in DHG were shown as follow:α1-9 N andα2-5 N in N-telopeptides,α1-87,α1-174,α1-930,α2-87,α2-174,α2-933 in triple helix domain,andα1-16 C in C-telopeptides.These hydroxylation and O-glycosylation were correlated with the formation and stability of collagen molecules and collagen fibrils.It is feasible for the collagens and peptides dissolving from deer skin collagen fibrils under high temperature and pressure decocting,high temperature and pressure also might destroy inter-molecular covalent cross-linking and help those glycol-peptides formations.The present study provided ideas and strategies for the in-depth investigation on DHG chemical constituents,and showed good theoretical significance and application value.