Mass spectrometrical identification of brain proteins including highly insoluble and transmembrane proteins.
Conventional two-dimensional electrophoresis (2DE) is the main technique used for protein profiling of tissues and cells, however separation of strongly acidic, basic or highly insoluble proteins is still limited. A series of methods have been proposed to cope with this problem and the use of discontinuous gel electrophoresis in an acidic buffer system using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride (16-BAC) with subsequent SDS-PAGE followed by mass spectrometry showed that results from 2DE can be complemented by this approach. It was the aim of this study to separate and identify proteins from whole mouse brain that were not demonstrated by 2DE. For this purpose samples were homogenised, soluble proteins were removed by ultracentrifugation and the water-insoluble pellet was resuspended in a mixture containing urea, 16-BAC, glycerol, pyronine Y and dithiothreitol. Electrophoresis was run in the presence of 16-BAC, the strip from the gel containing separated proteins was cut out and was re-run on SDS-PAGE. Protein spots were analyzed by MALDI-TOF-TOF mass spectrometry. One hundred and six individual proteins represented by 187 spots were unambiguously identified consisting of 42 proteins with predicted pI values of pI>8.0, 25 with a 6.0<pI<8.0 and 39 with a pI<6.0. Twelve proteins with transmembrane domains (ranging from 1 to 8) including channels and carriers were identified. The generated map revealed a series of important brain proteins that were not separated and identified previously. Therefore, this system may be relevant for protein chemical determination of channels and carriers independent of antibody availability and specificity. The fact that transmembrane, basic, acidic as well as hydrophobic proteins with a positive Gravy Index can be resolved warrants work on further improvement of this analytical tool.
Bierczynska-Krzysik
Anna
A
Department of Pediatrics, Medical University of Vienna, Waehringer Guertel 18, A-1090 Vienna, Austria.
Kang
Sung Ung
SU
Silberrring
Jerzy
J
Lubec
Gert
G
D000818:Animals; D001923:Brain Chemistry; D005260:Female; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins