Identification of transmembrane tryptic peptides of rhodopsin using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Identification of transmembrane tryptic peptides of rhodopsin using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
复制标题
使用基质辅助激光解吸/电离飞行时间质谱法鉴定视紫红质的跨膜胰蛋白酶肽。
DOI：
10.1002/pro.5560060408
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Jesaitis,AJ
中科院分区：
文献类型：
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作者：
Barnidge,DR;Dratz,EA;Sunner,J;Jesaitis,AJ
质谱法用于确定膜蛋白的整体拓扑的应用主要集中在不存在于脂质双层中的片段的质量测定上。在这项工作中，我们提出了准确的质量测定跨膜胰蛋白酶肽的牛视紫红质基质辅助激光解吸电离飞行时间质谱。确定疏水性跨膜肽的准确质量的能力将促进膜受体中配体结合位点的定位。它也将增加从脂质双层中消化的完整膜蛋白的跨膜区域的测定。用胰蛋白酶消化去污剂中的亲和纯化的视紫红质和视网膜杆膜中的视紫红质。使用反相高效液相色谱法在55°C下分离胰蛋白酶肽，洗涤剂辛基-β-葡糖苷在移动的相中。鉴定了视紫红质的六种跨膜胰蛋白酶肽中的四种，其质量范围为3，260 Da至6，528 Da。此外，通过MALDI MS鉴定了视紫红质N末端胰蛋白酶肽糖基化的异质性，分析前未修饰碳水化合物。
The application of mass spectrometry for determining the topography of integral membrane proteins has focused primarily on the mass determination of fragments that do not reside in the lipid bilayer. In this work, we present the accurate mass determination of transmembrane tryptic peptides of bovine rhodopsin using matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry. The ability to determine the accurate mass of hydrophobic transmembrane peptides will facilitate the mapping of ligand binding sites in membrane receptors. It will also augment the determination of membrane spanning regions from integral membrane proteins digested in lipid bilayers. Affinity‐purified rhodopsin in detergent and rhodopsin in retinal rod membranes were digested with trypsin. Tryptic peptides were separated using reverse‐phase, high‐performance liquid chromatography at 55°C with the detergent octyl‐β‐glucoside in the mobile phase. Four of the six transmembrane tryptic peptides of rhodopsin were identified, ranging in mass from 3,260 Da to 6,528 Da. The identities of the peptides were confirmed by Edman microsequencing. In addition, heterogeneity in the glycosylation of the N‐terminal tryptic peptide of rhodopsin was identified by MALDI MS, without modifying the carbohydrate prior to analysis.