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.
Keywords: mouse liver • plasma membrane • proteomics • immunoaffinity purification • MALDI-TOF-TOF • ESI-Q-TOF