Multidimensional Chromatography: a Powerful Tool for the Analysis of Membrane Proteins in Mouse Brain
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- 作者：Christiane Lohaus ; Andreas Nolte ; Martin Blü ; ggel ; Christian Scheer ; Joachim Klose ; Johan Gobom ; Andreas Schü ; ler ; Thomas Wiebringhaus ; Helmut E. Meyer ; Katrin Marcus
- 刊名：Journal of Proteome Research
- 出版年：2007
- 出版时间：January 2007
- 年：2007
- 卷：6
- 期：1
- 页码：105 - 113
- 全文大小：400K
- 年卷期：v.6,no.1(January 2007)
- ISSN：1535-3907
文摘
Understanding the function of membrane proteins is of fundamental importance due to their crucialroles in many cellular processes and their direct association with human disorders. However, theiranalysis poses a special challenge, largely due to their highly amphipathic nature. Until recently, analysesof proteomic samples mainly were performed by two-dimensional polyacrylamide gel electrophoresis(2D-PAGE), due to the unprecedented separation power of the technique. However, in conventional2D-PAGE membrane proteins are generally underrepresented due to their tendency to precipitate duringisoelectric focusing and their inefficient transfer from the first to the second dimension. As aconsequence, 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-basedmethods 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 digestswith 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 wasintroduced and a 3D-PAGE-HPLC analysis was performed. The results of these experiments arecompared with results of 2D-PAGE/matrix-assisted laser desorption ionization mass spectrometric(MALDI MS) analyses of the same samples. Features, challenges, advantages, and disadvantages ofthe respective systems are discussed. The brain (mouse and human) was chosen as the analyzed tissueas it is of high interest in medical and pharmaceutical research into neurological diseases such asmultiple sclerosis, stroke, Alzheimer's disease, and Parkinson's disease. The study is part of our ongoingresearch aimed at identifying new biomarkers for neurodegenerative diseases.