摘要
本研究通过对比三种常用的蛋白提取裂解液,建立适合少量卵丘颗粒细胞液质联用分析的蛋白提取方法。收集的卵细胞质内精子注入术患者的卵丘颗粒细胞,分别采用SDT、UED、RIPA裂解液提取卵丘颗粒细胞总蛋白,通过蛋白浓度测定检测蛋白提取效率,SDS-PAGE检测蛋白提取质量,并对酶解后的蛋白进行单针液质联用分析其表达谱,进而对蛋白的检测效果进行评估。蛋白浓度检测表明RIPA裂解液提取的卵丘颗粒细胞蛋白得率较UED裂解液高,蛋白凝胶条带则最为清晰,条带数量最多。液质联用检测发现UED裂解液提取的蛋白鉴定效率最高,RIPA裂解液提取的蛋白质谱峰图质量最佳,通过对鉴定蛋白亚细胞定位分析,发现RIPA裂解液对于膜蛋白鉴定效率上有明显优势,而UED裂解液可能有利于细胞核蛋白的检测。该研究表明三种方法中UED提取法更适合卵丘颗粒细胞的液质联用分析,为临床少量卵丘颗粒细胞的蛋白提取与液质联用分析提供方法依据,并为其他不易获得的少量样本的蛋白质组学提供参考。
In this study, we compared several lysates used for protein extraction in order to establish a protein extraction method suitable for the analysis of a small number of cumulus cells by LC-MS/MS. Cumulus cells were collected from intracyto-plasmic sperm injection patients and total protein was extracted by SDT, UED and RIPA lysates. Extraction efficiency was determined by protein concentration detection, extraction quality was determined by SDS-PAGE, and single injection of LC-MS/MS analysis was used to analyze the proteome profile. Protein concentration assay showed that the protein yield of cumulus cells was higher by RIPA lysate than UED lysate, and the protein gel bands were the most clear and the number of protein bands was the largest compared with the other lysates. LC-MS/MS analysis showed that the protein identification efficiency was highest when extracted from UED lysate, and the quality of protein spectrum derived from RIPA lysate was the best. Through subcellular localization analysis of proteome profile, we found that RIPA lysate had obvious advantages in the identification efficiency of membrane protein, and UED lysate may benefit for the identification of nucleus located proteins. The results showed that UED lysate was more suitable for the LC-MS/MS analysis of proteins from cumulus cells. This study provided a method for protein extraction and LC-MS/MS analysis of a small number of cumulus cells, and a reference for proteomics of other sample which was not easy to obtain.
作者
李楠
于凯
Li Nan;YuKai(Reproductive Medicine Center,Maternal and Child Health Hospital of Liuzhou,Liuzhou,545001;College of Animal Science and Technology,Guangxi University,Nanning,530004)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2021年第4期1871-1877,共7页
Genomics and Applied Biology
基金
广西自然科学基金(2015GXNSFBA139177)
广西卫计委自筹经费科研项目(Z2016560)
柳州市科学研究与计划开发计划资助课题(2016G020217)共同资助。
