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  Multidimensional chromatography: a powerful tool for the analysis of membrane proteins in mouse brain

Lohaus, C., Nolte, A., Blüggel, M., Scheer, C., Klose, J., Gobom, J., et al. (2007). Multidimensional chromatography: a powerful tool for the analysis of membrane proteins in mouse brain. Journal of Proteome Research, 6(1), 105-113. doi:10.1021/pr060247g.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-8298-5 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-8299-3
Genre: Journal Article
Alternative Title : J Proteome Res

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 Creators:
Lohaus, Christiane, Author
Nolte, Andreas, Author
Blüggel, Martin, Author
Scheer, Christian, Author
Klose, Joachim, Author
Gobom, Johan1, Author              
Schüler, Andreas, Author
Wiebringhaus, Thomas, Author
Meyer, Helmut E., Author
Marcus, Katrin, Author
Affiliations:
1Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_1433550              

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Free keywords: Multidimensional separation; Proteome analysis; LC-MS/MS data analysis; Membrane proteins
 Abstract: Understanding the function of membrane proteins is of fundamental importance due to their crucial roles in many cellular processes and their direct association with human disorders. However, their analysis poses a special challenge, largely due to their highly amphipathic nature. Until recently, analyses of proteomic samples mainly were performed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), due to the unprecedented separation power of the technique. However, in conventional 2D-PAGE membrane proteins are generally underrepresented due to their tendency to precipitate during isoelectric focusing and their inefficient transfer from the first to the second dimension. As a consequence, several other separation techniques, primarily based on liquid chromatography (LC), have been employed for analysis of this group of proteins. In the present study, different LC-based methods were compared for the analysis of crude protein extracts. One- and two-dimensional high-performance liquid chromatographic (1D- and 2D-HPLC) separations of brain protein tryptic digests with a predicted concentration range of up to 5 orders of magnitude were found to be insufficient, thus making a preceding fractionation step necessary. An additional protein separation step was introduced and a 3D-PAGE-HPLC analysis was performed. The results of these experiments are compared with results of 2D-PAGE/matrix-assisted laser desorption ionization mass spectrometric (MALDI MS) analyses of the same samples. Features, challenges, advantages, and disadvantages of the respective systems are discussed. The brain (mouse and human) was chosen as the analyzed tissue as it is of high interest in medical and pharmaceutical research into neurological diseases such as multiple sclerosis, stroke, Alzheimer's disease, and Parkinson's disease. The study is part of our ongoing research aimed at identifying new biomarkers for neurodegenerative diseases.

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Language(s): eng - English
 Dates: 2007-01
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Method: -
 Degree: -

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Title: Journal of Proteome Research
  Alternative Title : J Proteome Res
Source Genre: Journal
 Creator(s):
Affiliations:
Publ. Info: -
Pages: - Volume / Issue: 6 (1) Sequence Number: - Start / End Page: 105 - 113 Identifier: ISSN: 1535-3893