温和过甲酸氧化辅助酶解结合液质联用技术鉴定大鼠大脑皮层质膜蛋白质

Analysis of the Rat Cerebral Cortex Plasma Membrane Proteins by Mild Performic Acid Oxidation-assisted Digestion Coupled to LC-MS/MS

由于膜蛋白质尤其是内在膜蛋白的强疏水性,分析和鉴定质膜蛋白质仍然是以质谱为基础的蛋白质组学的方法中的一个难点.过甲酸氧化是一种应用广泛的打开二硫键的方法,温和的过甲酸试剂能完全的将半胱氨酸转化为半胱磺酸,将甲硫氨酸转化为甲硫氨酸砜,从而使目的蛋白更易溶于水介质.采用蔗糖密度梯度离心法纯化得到大鼠大脑皮层质膜,提取的质膜蛋白质经温和过甲酸氧化处理后经胰酶酶解消化得到肽段,利用LC-MS/MS对所得肽段进行质谱分析,采集的原始数据用Mascot软件进行库搜寻鉴定.此方法是研究质膜蛋白质的新方法,温和过甲酸氧化显示出很好的氧化效果却避免其它不利于鉴定的副反应.从大鼠大脑皮层膜提取物共鉴定出220种蛋白质,其中73种为整合膜蛋白,证明对质膜蛋白质直接进行温和过甲酸氧化然后酶解的方法辅助酶解可以有效的鉴定质膜蛋白质.

The low abundance and the hydrophobic nature of their transmembrane domains make them diffi-cult to analyze.Mild performic oxidation was explored as a method for sample pretreatment in membrane proteomic.Performic acid oxidation,a well-known method for cleave the disulfide bond can oxidizes methio-nine to methionine sulphone and cysteine to cysteic acid and make the oxidized protein more hydrophilic.The purified rat cerebral cortex plasma membrane proteins were oxidized by mild performic acid directly before hydrolysis by trypsin.The obtained peptides were analyzed by LC-MS/MS.The gentle performic acid oxidation assisted digestion methods shows good ability in membrane proteins identification.Using this method,219 of membrane proteins were identified from rat cerebral cortex membrane extracts,of which 73 are integral membrane proteins.The results proved the method allows large-scale detection and identification of membrane proteins.