[Analysis of mouse liver membrane proteins using multidimensional ion exchange chromatography and tandem mass spectrometry].
The analysis of membrane proteins is still a technical obstacle in proteomic investigation. A fundamental question is how to allow the hydrophobic proteins fully solubilizing in a proper solvent environment. We propose that the denatured membrane proteins in high denaturant solution are fully ionized and separated through ion exchange chromatography. The membrane proteins prepared from a mouse liver were dissolved in 4 mol/L urea, 20 mmol/L Tris-HCl buffer (pH 9.0), and loaded onto a tandem chromatography coupled with Q-Sepharose FF and Sephacryl S-200HR. With a linear NaCl gradient elution, the bound proteins were eluted and collected followed by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to further separate the eluted proteins. The protein bound on SDS-PAGE were excised and in-gel digested by trypsin, while the digested peptides were delivered to reversed-phase high performance liquid chromatography (HPLC) and ion-trap mass spectrometry for the peptide identifications. Of a total of 392 proteins identified, 306 were membrane proteins or membrane associated proteins reported by literature. Based on the calculation of hydrophobicity, the GRAVY (grand average of hydropathicity) scores of 83 proteins are over or equal to 0.00. Taking all the evidence, we have established an effective approach which is feasible in the investigation towards mouse liver membrane proteomics.
Wang
Zhuowei
Z
Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 101318, China.
Peng
Fuli
F
Wang
Yuan
Y
Tong
Wei
W
Ren
Yan
Y
Xu
Ningzhi
N
Liu
Siqi
S
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005260:Female; D008099:Liver / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
D008565:Membrane Proteins