title: Prefractionation and separation by C8 stationary phase: effective strategies for integral membrane proteins analysis.
abstract: Analysis of integral membrane proteins (IMPs) presents great challenges due to their hydrophobic nature. Recently, much attention has been paid to improve the solubilization of IMPs. However, besides that, the separation of hydrophobic peptides with high recovery is also a dominating factor, but with rare report. Here, the prefractionation of the digests by reverse phase trap column during desalting was presented to efficiently decrease the complexity of samples, with the identified hydrophobic peptides and IMPs increased by more than 43%. Furthermore, the effect of C18 and C8 stationary phases on the separation of membrane protein digests was studied. A total of 301 proteins (536 peptides) with C18 stationary phase and 398 proteins (703 peptides) with C8 stationary phase were identified by μRPLC-ESI-MS/MS using an LCQ instrument in duplicate runs, with false discovery rate (FDR) less than 5% at peptide level. In addition, with C8 stationary phase, the number of identified hydrophobic peptides and IMPs was obviously improved by 29% and 20%, respectively, compared with that identified by C18 stationary phase, indicating that the polarity of stationary phase has evident effect on the analysis of membrane protein digests. All these results show that the prefractionation by reverse phase trap column during desalting and the separation by C8 stationary phases could facilitate the efficient identification of IMPs.
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian 116023, China.
authors.forename: Qun
authors.initials: Q
authors.lastname: Zhao
authors.forename: Liangliang
authors.initials: L
authors.lastname: Sun
authors.forename: Yu
authors.initials: Y
authors.lastname: Liang
authors.forename: Qi
authors.initials: Q
authors.lastname: Wu
authors.forename: Huiming
authors.initials: H
authors.lastname: Yuan
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liang
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.forename: Yukui
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002417:Cattle; D005591:Chemical Fractionation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D006624:Hippocampus / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D008297:Male; D008565:Membrane Proteins / Q000032:analysis*; D010455:Peptides / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D010455:Peptides; D014357:Trypsin