title: Development and application of a two-phase, on-membrane digestion method in the analysis of membrane proteome.
abstract: Analysis of membrane proteins, particularly integral membrane proteins, still presents a great challenge due to their poor water solubility and low abundance though much effort has been devoted to the solubilization and enrichment of the protein class. In this paper, a two-phase, on-membrane digestion method was developed and applied in the analysis of rat liver membrane proteome. The two-phase system was constituted by mixing n-butanol and 25 mM NH4HCO3. Comparative experiments indicated that the proteins on membranes could be digested in the two-phase system more efficiently than in both 60% methanol and 25 mM NH4HCO3 solutions under the same conditions, thereby improving the identification of the membrane proteins. When the established two-phase system and CapLC-MS/MS was used to analyze rat liver membrane proteome, a total of 411 membrane proteins were identified, more than 80% of which were transmembrane proteins with 1-12 mapped transmembrane domains (TMDs). Because of its extraction and dissolution actions, the two-phase on-membrane digestion system we developed could efficiently improve the digestion and removal of adsorbed nonmembrane proteins, and remarkably increase the number and coverage of identified membrane proteins, particularly the transmembrane proteins. Using our procedure to identify a complementary protein set from all fractions of the two-phase system could achieve a higher coverage of the membrane proteome.
authors.affiliation: College of Life Sciences, Hunan Normal University, Changsha 410081, P. R. China.
authors.forename: Jian
authors.initials: J
authors.lastname: Zhou
authors.forename: Yong
authors.initials: Y
authors.lastname: Lin
authors.forename: Xingcan
authors.initials: X
authors.lastname: Deng
authors.forename: Jianying
authors.initials: J
authors.lastname: Shen
authors.forename: Quanyuan
authors.initials: Q
authors.lastname: He
authors.forename: Ping
authors.initials: P
authors.lastname: Chen
authors.forename: Xianchun
authors.initials: X
authors.lastname: Wang
authors.forename: Songping
authors.initials: S
authors.lastname: Liang
mesh_terms: D020001:1-Butanol / Q000737:chemistry; D000818:Animals; D001639:Bicarbonates / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods; D006868:Hydrolysis; D008099:Liver / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D000432:Methanol / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
chemical_list: D001639:Bicarbonates; D008565:Membrane Proteins; C027043:ammonium bicarbonate; D020001:1-Butanol; D014357:Trypsin; D000432:Methanol