流水浸泡槽插入0.5mLProteinLoBind管内(EppendorgAG,Habburg,Derland)用甘露穿透底部2mL质谱管并最大速度离心机5分钟Posa等初始描述程序
18号并允许提取流泪催泪液存储于-80摄氏度直到NLC-MS/MS进一步分析
2微值LC-MS/MS稀释液8m尿素/100mTris样本填充达20ml+6xLaemli缓冲并煮5分钟95摄氏度后加载12.5%SDS-PAGE凝胶样本开发成凝胶约1.5cmProteins与Coomassie蓝色染色,每条车道切成五波段等大gel带切成小立方块,转成1.5ml聚丙烯反应小瓶并用100mL20%乙醇湿化存储4°C直至消化
关于NLC-MS/MS分析,20个样本随机整理以控制样本处理偏差Proteins用凝胶消化,别处描述
19号sm/s分析由EASY-nLC1000色谱相加快速高频质谱仪组成的工具搭建Peptides嵌入AcclaimC18PepMap100前列(5m,100+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++检测m/z400至1400的分辨率为60 000(m/z250),自动增益控制目标为1E06,最大离子注入时间为50ms顶值15数据依存分解法应用如下设置:前端离子宽1.6m/z、分辨率1550、AGC1E05、最大离子时间110ms、电荷加2+7+ions、Peptide匹配和动态排除20秒
质谱数据用maxQuant/Andromeda1.6.1.0处理,使用默认设置检测峰值,严格切分规则允许最多三次漏分,对metionine可变氧化化,蛋白N-termini退缩,对asqine和drome串行匹配使用时相邻凝胶分数间保留0.7分钟,样本间则不使用SwissProt人类蛋白序列数据库(2019_02版)用于解释碎片谱,初始质容度分别为先质10ppm和碎片离子20ppm接受蛋白质识别法时,每组蛋白至少两片薄片以1%假发现率截取跨样本解析最大量计算蛋白类LFQ强度中值LFQ强度用作调试的临界值:如果所有样本中值LFQ质组小于样本中子值LFQ值,则对应样本中蛋白度被视为上调LFQ类中位数高或等值蛋白类中值,则相关样本中蛋白调低