human 293 cells PEDF LC-MS/MS - Adenoviral E4 gene stimulates secretion of pigmental epithelium derived factor (PEDF) that maintains long-term survival of human glomerulus-derived endothelial cells

Experiment was performed on 293 cells, details are presented in publication: Adenoviral E4 gene stimulates secretion of PEDF that maintains long-term survival of human glomerulus-derived endothelial cells Marina Jerebtsova‡§*, Namita Kumari‖, Yuri Obuhkov‖, and Sergei Nekhai‖†* (to be published in Molecular and Cellular Proteomics), please see more details in paper Workflow HPLC on PicoFrit C18 column, gradient 60 min from 2% to 30% ACN Nanospray 2 kV, flow rate 300 nL/min LTQ Orbitrap MS. Data dependent scan FT MS + 3 FT MS/MS. A blank water run was performed after each sample run Each 24 hours setup performance was tested on Apomyoglobin sample, 12 fmol. Coverage was 39-79 % Search is made by SEQUEST on human database

本实验于293细胞（293 cells）上开展，详细信息见于待发表于《Molecular and Cellular Proteomics》（分子与细胞蛋白质组学）的论文：《腺病毒E4基因刺激色素上皮衍生因子（PEDF）分泌以维持人肾小球源内皮细胞的长期存活》，作者为Marina Jerebtsova‡§*、Namita Kumari‖、Yuri Obuhkov‖及Sergei Nekhai‖†*，更多实验细节可查阅该论文。实验流程如下：采用PicoFrit C18色谱柱进行高效液相色谱（HPLC）分离，以60分钟梯度洗脱，流动相乙腈（ACN）比例从2%升至30%；采用纳升电喷雾电离，电压2 kV，流速300 nL/min；使用LTQ Orbitrap质谱仪进行数据采集，采用数据依赖型扫描模式：傅里叶变换质谱（FT MS）搭配3次傅里叶变换串联质谱（FT MS/MS）。每完成一组样本分析后，均进行一次空白水溶液进样以清除系统残留。每日均采用12 fmol的脱辅基肌红蛋白（Apomyoglobin）标准品对实验装置性能进行验证，其肽段序列覆盖度可达39%~79%。质谱数据检索采用SEQUEST算法，检索库为人类蛋白质组数据库。