Sample preparation for the analysis of membrane proteomes by mass spectrometry

Abstract

The low abundance and highly hydrophobic nature of most membrane proteins make their analysis more difficult than that for common soluble proteins. Successful membrane protein identification is largely dependent on the sample preparation including the enrichment and dissolution of the membrane proteins. A series of conventional and newly developed methods has been applied to the enrichment of low-abundance membrane proteins at membrane and/or protein levels and to the dissolution of hydrophobic membrane proteins. However, all the existing methods have inherent advantages and limitations. Up to now, there has been no unique method that can universally be employed to solve all the problems and more efforts are needed in improving sample preparation for the analysis of membrane proteomes.

Special Enrichment Strategies Greatly Increase the Efficiency of Missing Proteins Identification from Regular Proteome Samples

“…For mining MPs from the membrane proteome, membrane proteins were prepared from MHCC97L and HCCLM6 cell lines using ultracentrifugation as described previously. , Briefly, the cell homogenate was centrifuged at 9600 g for 15 min at 4 °C. Then, the resulting supernatant was centrifuged at 120 000 g for 80 min at 4 °C.…”

Protein oxidation - Formation mechanisms, detection and relevance as biomarkers in human diseases

“…Extraction steps might be applied to reduce the number of interfering compounds. Simultaneously, this leads to enrichment of proteins-of-interest which might be of importance for low abundant proteins [ 76 ]. Chemicals used for protein extraction should be chosen with caution since some of them can interfere with the protein oxidation analysis.…”

A Primer on Concepts and Applications of Proteomics in Neuroscience

“…Given the low abundance and the amphipathicity of most membraneassociated proteins, special care has been taken in selecting proper lysis conditions to ensure proper solubilization of membrane proteins. Several studies have addressed the extraction and digestion properties of proteins associated with bilayer membranes in order to enhance their identification (Shevchenko et al, 2012;Wang and Liang, 2012). For instance, several membrane fractionation steps have been used to characterize the microglia membrane proteome, and this approach can even be combined with the selective enrichment of glycated peptides for glycoproteome analysis (Han et al, 2014b).…”