title: Analysis of membrane and hydrophilic proteins simultaneously derived from the mouse brain using cloud-point extraction.
abstract: In this study, a temperature-induced phase fractionation known as cloud-point extraction (CPE) with the non-ionic surfactant Triton X-114 was used to simultaneously extract, concentrate, and fractionate hydrophobic and hydrophilic proteins from mouse brain tissue. Two bottom-up proteomic techniques were used to comprehensively identify the extracted proteins. The first "shotgun"-based approach included tryptic digestion of the proteins followed by reversed-phase nanoliquid chromatography (RP-nanoLC) in combination with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). In the second approach, the extracted intact proteins were first separated by one-dimensional (1D) gel electrophoresis and then in-gel digested with trypsin and analyzed with nanoLC-MS/MS. In total, 1,825 proteins were unambiguously identified and the percentage of membrane proteins was 26% which is at the reported genome expression levels of 20-30%. The protein overlap between the two approaches was high. The majority (77%) of the identifications in the first approach was also found by the second method. The protein overlap between the CPE-extracted hydrophilic and hydrophobic fractions was rather small (16-23%) for both methods, which indicates a good phase separation. A quantitative evaluation of the CPE with iTRAQ labeling and nanoLC-ESI-MS/MS analysis gave iTRAQ ratios at the expected levels and an overall variation of the entire method at 17-31%. The results indicate very reproducible sample preparation and analysis methods that readily can be applied on large-scale sample sets.
authors.affiliation: Department of Physical and Analytical Chemistry, Uppsala University, Sweden. magnus.wetterhall@kemi.uu.se
authors.forename: Magnus
authors.initials: M
authors.lastname: Wetterhall
authors.forename: Ganna
authors.initials: G
authors.lastname: Shevchenko
authors.forename: Konstantin
authors.initials: K
authors.lastname: Artemenko
authors.forename: Marcus O D
authors.initials: MO
authors.lastname: Sjödin
authors.forename: Jonas
authors.initials: J
authors.lastname: Bergquist
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D005591:Chemical Fractionation / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000302:isolation & purification*; D051379:Mice; D017830:Octoxynol; D011092:Polyethylene Glycols; D011506:Proteins / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D013696:Temperature
chemical_list: D008565:Membrane Proteins; D011506:Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40