A detergent- and cyanogen bromide-free method for integral membrane proteomics: application to Halobacterium purple membranes and the human epidermal membrane proteome.
A simple and rapid method for characterizing hydrophobic integral membrane proteins and its utility for membrane proteomics using microcapillary liquid chromatography coupled on-line with tandem mass spectrometry (microLC-MS/MS) is described. The present technique does not rely on the use of detergents, strong organic acids or cyanogen bromide-mediated proteolysis. A buffered solution of 60% methanol was used to extract, solubilize, and tryptically digest proteins within a preparation of Halobacterium (H.) halobium purple membranes. Analysis of the digested purple membrane proteins by microLC-MS/MS resulted in the identification of all the predicted tryptic peptides of bacteriorhodopsin, including those that are known to be post-translationally modified. In addition, 40 proteins from the purple membrane preparation were also identified, of which 80% are predicted to contain between 1 and 16 transmembrane domains. To evaluate the general applicability of the method, the same extraction, solubilization, and digestion conditions were applied to a plasma membrane fraction prepared from human epidermal sheets. A total of 117 proteins was identified in a single microLC-MS/MS analysis, of which 55% are known to be integral or associated with the plasma membrane. Due to its simplicity, efficiency, and absence of MS interfering compounds, this technique can be used for the characterization of other integral membrane proteins and may be concomitantly applied for the analysis of membrane protein complexes or large-scale proteomic studies of different membrane samples.
Blonder
Josip
J
SAIC-Frederick Inc., Laboratory of Proteomics and Analytical Technologies Mass Spectrometry Center, National Cancer Institute at Frederick, Frederick, MD 21702-1201, USA.
Conrads
Thomas P
TP
Yu
Li-Rong
LR
Terunuma
Atsushi
A
Janini
George M
GM
Issaq
Haleem J
HJ
Vogel
Jonathan C
JC
Veenstra
Timothy D
TD
D001436:Bacteriorhodopsins / Q000737:chemistry; D002462:Cell Membrane / Q000737:chemistry; D003488:Cyanogen Bromide; D003902:Detergents; D004817:Epidermis / Q000737:chemistry; D006217:Halobacterium / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D018717:Purple Membrane / Q000737:chemistry*
D003902:Detergents; D008565:Membrane Proteins; D001436:Bacteriorhodopsins; D003488:Cyanogen Bromide