title: Neuroproteomic profiling of human brain tissue using multidimensional separation techniques and selective enrichment of membrane proteins.
abstract: Hydrophobic membrane proteins (MPs) occupy a unique niche in the brain proteome research due to their important physiological roles. Therefore, the extraction, separation, and identification of MPs are of great interest in proteomic analysis. We applied various proteomic techniques to enrich, separate, and analyze the human brain proteome, including membrane proteome. Temperature-induced phase fractionation with the nonionic surfactant Triton X-114 was used to simultaneously extract, separate, and concentrate low abundant hydrophobic and high abundant hydrophilic proteins from human brain tissue. The extracted and delipidated proteins were analyzed by two-dimensional gel electrophoresis (2DE). Approximately 600 spots were detected in the gels. In-solution digestion was performed on 3 kDa spin filters. Tryptic peptides were separated using RP nano-LC and analyzed using two different high performance mass spectrometers, linear ion trap-Fourier transform and a linear ion trap-Orbitrap to reveal the low abundant MPs. In total, 837 and 780 unique proteins were identified by using linear ion trap-Fourier transform and linear ion trap-Orbitrap mass spectrometers, respectively. More than 29% of the identified proteins were classified as MPs with significant biological functions such as ion channels and transporters. Our study establishes a simple and rapid shotgun approach for the characterization of the brain proteome, and allows comprehensive analysis of brain membrane proteomes.
authors.affiliation: Analytical Chemistry, Department of Chemistry-Biomedical Center and Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Sravani
authors.initials: S
authors.lastname: Musunuri
authors.forename: Ganna
authors.initials: G
authors.lastname: Shevchenko
authors.forename: Jonas
authors.initials: J
authors.lastname: Bergquist
mesh_terms: D001921:Brain / Q000378:metabolism*; D001923:Brain Chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D020543:Proteome / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*
chemical_list: D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D020543:Proteome