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Analysis of membrane and hydrophilic proteins simultaneously derived from the mouse brain using cloud-point extraction

Authors :
Jonas Bergquist
Marcus O.D. Sjödin
Konstantin A. Artemenko
Magnus Wetterhall
Ganna Shevchenko
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.

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