Reproducible method to enrich membrane proteins with high purity and high yield for an LC-MS/MS approach in quantitative membrane proteomics.

ABSTRACT: The proportionately low abundance of membrane proteins hampers their proteomic analysis, especially for a quantitative LC-MS/MS approach. To overcome this limitation, a method was developed that consists of one cell disruption step in a hypotonic reagent using liquid nitrogen, one isolation step using a low speed centrifugation, and three wash steps using high speed centrifugation. Pellets contained plasma, nuclear, and mitochondrial membranes, including their integral, peripheral, and anchored membrane proteins. The reproducibility of this method was verified by protein assay of four separate experiments with a CV of 7.7%, and by comparative LC-MS/MS label-free quantification of individual proteins between two experiments with 99% of the quantified proteins having a CV ≤30%. Western blot

SUBMITTER: Lai X 

PROVIDER: S-EPMC3749515 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature