Coupling formic acid assisted solubilization and online immobilized pepsin digestion with strong cation exchange and microflow reversed-phase liquid chromatography with electrospray ionization tandem mass spectrometry for integral membrane proteome analysis.
In this study, a facile system for membrane proteome profiling was established, in which membrane proteins were solubilized by formic acid, online digested by a pepsin-based immobilized enzyme reactor (pepsin-IMER), and analyzed by strong cation exchange and microflow reversed-phase liquid chromatography with electrospray ionization tandem mass spectrometry (SCX-μRPLC-ESI-MS/MS). Under optimized conditions, such a system showed excellent compatibility between all crucial steps and was successfully applied for analyzing integral membrane proteins extracted from rat liver microsomes. Out of the 235 unique proteins positively identified, 39% (91/235) were annotated as membrane proteins with one or more transmembrane domains (TMDs). It is anticipated that the efficient sample treatment and the relevant online analytical system might provide a promising tool for automated and comprehensive profiling of membrane proteomes.
Junfeng
J
Ma
Chunyan
C
Hou
Liangliang
L
Sun
Dingyin
D
Tao
Yanyan
Y
Zhang
Yichu
Y
Shan
Zhen
Z
Liang
Lihua
L
Zhang
Ling
L
Yang
Yukui
Y
Zhang
D000818:Animals; D002852:Chromatography, Ion Exchange / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D005561:Formates / Q000737:chemistry*; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008862:Microsomes, Liver / Q000378:metabolism; D010434:Pepsin A / Q000737:chemistry / Q000378:metabolism*; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D005561:Formates; D055767:Immobilized Proteins; D008565:Membrane Proteins; D020543:Proteome; C030544:formic acid; D010434:Pepsin A