Proteomics is potentially a powerful technology for elucidating brain function and neurodegenerative diseases. So far, the brain proteome has generally been analyzed by two-dimensional gel electrophoresis, which usually leads to the complete absence of membrane proteins. We describe a proteomic approach for profiling of plasma membrane proteins from mouse brain. The procedure consists of a novel method for extraction and fractionation of membranes, on-membrane digestion, diagonal separation of peptides, and high-sensitivity analysis by advanced MS. Breaking with the classical plasma membrane fractionation approach, membranes are isolated without cell compartment isolation, by stepwise depletion of nonmembrane molecules from entire tissue homogenate by high-salt, carbonate, and urea washes followed by treatment of the membranes with sublytic concentrations of digitonin. Plasma membrane is further enriched by of density gradient fractionation and protein digested on-membrane by endoproteinase Lys-C. Released peptides are separated, fractions digested by trypsin, and analyzed by LC-MS/MS. In single experiments, the developed technology enabled identification of 862 proteins from 150 mg of mouse brain cortex. Further development and miniaturization allowed analysis of 15 mg of hippocampus, revealing 1,685 proteins. More that 60% of the identified proteins are membrane proteins, including several classes of ion channels and neurotransmitter receptors. Our work now allows in-depth study of brain membrane proteomes, such as of mouse models of neurological disease.
A novel isotopically labeled cysteine-tagging and complexity-reducing reagent, called HysTag, has been synthesized and used for quantitative proteomics of proteins from enriched plasma membrane preparations from mouse fore-and hindbrain. The reagent is a 10-mer derivatized peptide, H 2 N-(His) 6 -Ala-Arg-Ala-Cys(2-thiopyridyl disulfide)-CO 2 H, which consists of four functional elements: i) an affinity ligand (His 6 -tag), ii) a tryptic cleavage site (-Arg-Ala-), iii) Ala-9 residue that contains four (d 4 ) or no (d 0 ) deuterium atoms, and iv) a thiol-reactive group (2-thiopyridyl disulfide). For differential analysis cysteine residues in the compared samples are modified using either (d 4 ) or (d 0 ) reagent. The HysTag peptide is preserved in Lys-C digestion of proteins and allows chargebased selection of cysteine-containing peptides, whereas subsequent tryptic digestion reduces the labeling group to a di-peptide, which does not hinder effective fragmentation. Furthermore, we found that tagged peptides containing Ala-d 4 co-elute with their d 0 -labeled counterparts. To demonstrate effectiveness of the reagent, a differential analysis of mouse forebrain versus hindbrain plasma membranes was performed. Enriched plasma membrane fractions were partially denatured, reduced, and reacted with the reagent. Digestion with endoproteinase Lys-C was carried out on nonsolubilized membranes. The membranes were sedimented by ultra centrifugation, and the tagged peptides were isolated by Ni 2؉ affinity or cationexchange chromatography. Finally, the tagged peptides were cleaved with trypsin to release the histidine tag (residues 1-8 of the reagent) followed by liquid chromatography tandem mass spectroscopy for relative protein quantification and identification. A total of 355 unique proteins were identified, among which 281 could be quantified. Among a large majority of proteins with ratios close to one, a few proteins with significant quantitative changes were retrieved. The HysTag offers advantages compared with the isotope-coded affinity tag reagent, because the HysTag reagent is easy to synthesize, economical due to use of deuterium instead of 13 C isotope label, and allows robust purification and flexibility through the affinity tag, which can be extended to different peptide functionalities.Molecular & Cellular Proteomics 3: 82-92, 2004.
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