Immunoaffinity purification of plasma membrane with secondary antibody superparamagnetic beads for proteomic analysis.
Plasma membrane (PM) has very important roles in cell-cell interaction and signal transduction, and it has been extensively targeted for drug design. A major prerequisite for the analysis of PM proteome is the preparation of PM with high purity. Density gradient centrifugation has been commonly employed to isolate PM, but it often occurred with contamination of internal membrane. Here we describe a method for plasma membrane purification using second antibody superparamagnetic beads that combines subcellular fractionation and immunoisolation strategies. Four methods of immunoaffinity were compared, and the variation of crude plasma membrane (CPM), superparamagnetic beads, and antibodies was studied. The optimized method and the number of CPM, beads, and antibodies suitable for proteome analysis were obtained. The PM of mouse liver was enriched 3-fold in comparison with the density gradient centrifugation method, and contamination from mitochondria was reduced 2-fold. The PM protein bands were extracted and trypsin-digested, and the resulting peptides were resolved and characterized by MALDI-TOF-TOF and ESI-Q-TOF, respectively. Mascot software was used to analyze the data against IPI-mouse protein database. Nonredundant proteins (248) were identified, of which 67% are PM or PM-related proteins. No endoplasmic reticulum (ER) or nuclear proteins were identified according to the GO annotation in the optimized method. Our protocol represents a simple, economic, and reproducible tool for the proteomic characterization of liver plasma membrane.
Zhang
Lijun
L
Key Laboratory of Protein Chemistry and Developmental Biology of National Education Committee, College of Life Science, Hunan Normal University, Changsha 410081, PR China.
Wang
Xi'e
X
Peng
Xia
X
Wei
Yingjuan
Y
Cao
Rui
R
Liu
Zhen
Z
Xiong
Jixian
J
Ying
Xingfeng
X
Chen
Ping
P
Liang
Songping
S
D000818:Animals; D002450:Cell Communication; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D030562:Databases, Protein; D018189:Immunomagnetic Separation / Q000379:methods*; D008099:Liver / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014357:Trypsin / Q000494:pharmacology
D014357:Trypsin