Detergent-based but gel-free method allows identification of several hundred membrane proteins in single LC-MS runs.
Detergents are indispensable solubilizing agents in the purification and analysis of membrane proteins. For mass spectrometric identification of proteins, it is essential that detergents are removed prior to analysis, necessitating an in-gel digestion step. Here, we report a procedure that allows use of detergents and in-solution digestion of proteins. Crude membrane preparations from mouse brain were solubilized with Triton X-100, CHAPS, or SDS, and the detergents were depleted from the membrane proteins using a desalting column equilibrated with 8 M urea. Following digestion with endoproteinase Lys-C, the resulting peptides were analyzed by LC-MS/MS on Linear ion trap-Orbitrap instrument. Applying stringent identification criteria, in single-LC-MS-runs, 1059 +/- 108 proteins, including 797 +/- 43 membrane proteins, were mapped from mouse brain. The identified proteins represented a broad spectrum of neurotransmitter receptors and other ion channels. The general applicability of the method is demonstrated by profiling of membrane proteins from four other mouse organs. Single-run analyses of eye, liver, spleen, and skeletal muscle allowed identification of 522 +/- 9, 610 +/- 7, 777 +/- 8, and 307 +/- 7 membrane proteins. Our results demonstrate that membrane proteins can be analyzed as efficiently as soluble proteins.
Department of Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry, Martinsried, Germany.
Nagarjuna
N
Nagaraj
Aiping
A
Lu
Matthias
M
Mann
Jacek R
JR
Wiśniewski
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D019295:Computational Biology / Q000379:methods; D003902:Detergents / Q000032:analysis*; D005782:Gels / Q000032:analysis*; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D051379:Mice
D003902:Detergents; D005782:Gels; D008565:Membrane Proteins