Cysteinyl-tagging of integral membrane proteins for proteomic analysis using liquid chromatography-tandem mass spectrometry.
Membrane proteomic analysis is of considerable interest due to the role of receptors, ion channels, and membrane-associated proteins that are critical components in cellular control and differentiation. Consequently, proteomic investigations of membrane proteins under a variety of conditions and stimuli are being conducted. Although abundant and biologically significant, large-scale proteomic analysis of highly hydrophobic integral membrane proteins containing multiple transmembrane domains (TMDs) is more difficult and requires alternative methods than those routinely used for whole-cell proteomic studies. This chapter contains a method for extraction, solubilization, cysteinyl-labeling, proteolysis, and identification of hydrophobic integral membrane proteins for large-scale proteomic analysis using liquid chromatography-tandem mass spectrometry (LC/MS/MS). Application of this method enables proteome-wide identification of integral membrane proteins from both bacterial and plant tissues. The method is also amenable to quantifying integral membrane protein expression and posttranslational modifications using isotopically enriched media or various stable isotope-labeling and/or affinity isolation reagents such as iTRAQ and cICAT. Since the protocol can easily be extended to various cell and tissue types, we anticipate that the method will be of interest to those who are trying to characterize the membrane proteome and gain some insight regarding the role of receptors, ion channels, and other membrane proteins involved in signal transduction and cellular differentiation pathways.
Mitra
Srijeet K
SK
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, USA.
Goshe
Michael B
MB
D017360:Arabidopsis / Q000737:chemistry; D001419:Bacteria / Q000737:chemistry; D001426:Bacterial Proteins / Q000032:analysis* / Q000378:metabolism; D019921:Biotinylation; D002853:Chromatography, Liquid; D003545:Cysteine / Q000737:chemistry; D007553:Isotope Labeling; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D010940:Plant Proteins / Q000032:analysis* / Q000378:metabolism; D010944:Plants / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012995:Solubility; D053719:Tandem Mass Spectrometry
D001426:Bacterial Proteins; D008565:Membrane Proteins; D010940:Plant Proteins; D003545:Cysteine