Evaluation and optimization of removal of an acid-insoluble surfactant for shotgun analysis of membrane proteome.
Due to its compatibility with protease activity at high concentration, sodium deoxycholate (SDC) can be used to effectively improve the solubilization and enzymolysis of membrane proteins and has received increasing attention in the field of membrane proteome analysis in recent years. SDC can be removed from digests by means of acidification followed by centrifugation (i.e. acid precipitation, AP) or extraction with ethyl acetate (i.e. phase transfer, PT) so as not to interfere with the downstream analyses like LC-MS/MS. In this study, the two strategies were systematically evaluated, compared and optimized. The results of the study demonstrated that both of the AP and PT strategies led to a certain amount of tryptic peptides being lost, and in PT strategy even more peptides were lost during SDC removal process. However, the lost peptides could be mostly recovered by washing the pellet and solid content produced during AP and PT, respectively. By recovering the lost peptides, the identification efficiency of proteins, especially transmembrane and low abundance ones, was significantly improved. Comparatively, after optimization by recovering the lost peptides, AP strategy was superior to PT strategy because the former not only could achieve the comparable identification efficiency with the latter but also was more economical, safer and easier to operate than the latter.
Lin
Yong
Y
Key Laboratory of Protein Chemistry and Developmental Biology of National Education Committee, College of Life Sciences, Hunan Normal University, Changsha, P. R. China.
Liu
Yi
Y
Li
Jianjun
J
Zhao
Yan
Y
He
Quanze
Q
Han
Wenjun
W
Chen
Ping
P
Wang
Xianchun
X
Liang
Songping
S
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D019295:Computational Biology; D003840:Deoxycholic Acid; D008099:Liver / Q000737:chemistry; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D020543:Proteome / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D012995:Solubility; D013501:Surface-Active Agents
D008565:Membrane Proteins; D010455:Peptides; D020543:Proteome; D013501:Surface-Active Agents; D003840:Deoxycholic Acid