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Analysis of membrane and hydrophilic proteins simultaneously derived from the mouse brain using cloud-point extraction
- Source :
- Analytical and Bioanalytical Chemistry. 400:2827-2836
- Publication Year :
- 2011
- Publisher :
- Springer Science and Business Media LLC, 2011.
-
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.
- Subjects :
- Proteomics
Spectrometry, Mass, Electrospray Ionization
Octoxynol
Electrospray ionization
Fractionation
Chemical Fractionation
Tandem mass spectrometry
Mass spectrometry
Biochemistry
Polyethylene Glycols
Analytical Chemistry
Mice
Pulmonary surfactant
Tandem Mass Spectrometry
Animals
Brain Chemistry
Gel electrophoresis
Chromatography
Chemistry
Temperature
Membrane Proteins
Proteins
Reproducibility of Results
Membrane protein
Hydrophobic and Hydrophilic Interactions
Subjects
Details
- ISSN :
- 16182650 and 16182642
- Volume :
- 400
- Database :
- OpenAIRE
- Journal :
- Analytical and Bioanalytical Chemistry
- Accession number :
- edsair.doi.dedup.....8509d2f5b3f165a978658b88d249dda7
- Full Text :
- https://doi.org/10.1007/s00216-011-5037-9