Enrichment and preparation of plasma membrane proteins from Arabidopsis thaliana for global proteomic analysis using liquid chromatography-tandem mass spectrometry.
The plasma membrane proteins are critical components in cellular control and differentiation and thus are of special interest to those studying signal transduction mechanisms in all organisms. When conducting proteomic studies on membrane components of cells and tissues, the complexity is not simply confined to the large number of proteins present in the sample but also to the highly hydrophobic nature of membrane proteins containing multiple transmembrane domains. Consequently, these proteins are more difficult to analyze by mass spectrometry, particularly if protein sequence coverage is to be established. This chapter contains a method for extraction, solubilization, alkylation, proteolysis, and identification of hydrophobic integral plasma membrane proteins for large-scale proteomic analysis using strong cation exchange chromatography (SCXC) and liquid chromatography-tandem mass spectrometry (LC/MS/MS). In our approach, microsomes are isolated from plant tissue and then subjected to a two-phase extraction procedure to enrich for plasma membranes. Proteins are extracted and solubilized from the membrane using a methanol-aqueous buffer system that allows for effective reduction, cysteinyl alkylation, and tryptic digestion for subsequent SCXC-LC/MS/MS analysis. Our protocol is also amenable to isotope labeling methods to quantify integral membrane protein expression and post-translational modifications. In addition to plants, the method can be applied to other systems quite readily; thus, we anticipate that it will be of general interest to those characterizing plasma membrane proteins of any organism.
Mitra
Srijeet K
SK
Department of Horicultural Science, North Carolina State University, Raleigh, NC 27695-7609, USA.
Clouse
Steven D
SD
Goshe
Michael B
MB
D017360:Arabidopsis / Q000737:chemistry*; D029681:Arabidopsis Proteins / Q000032:analysis*; D002462:Cell Membrane / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D008565:Membrane Proteins / Q000032:analysis*; D008861:Microsomes / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
D029681:Arabidopsis Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments