Isolation and identification of the plasma membrane-associated intracellular protein reggie-2 from goldfish brain by chromatography and Fourier-transform ion cyclotron resonance mass spectrometry
| Autor: | Michael Przybylski, Sebastian H.J. Bauer, Claudia A. O. Stuermer, Marianne Wiechers, Kai Bruns |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
Fish Proteins
Protein mass spectrometry Microdomains Molecular Sequence Data Biophysics Nerve Tissue Proteins Mass spectrometry Biochemistry Fourier transform ion cyclotron resonance Antibodies Western blot Affinity chromatography ddc:570 Goldfish Spectroscopy Fourier Transform Infrared medicine Animals Amino Acid Sequence Phosphorylation Reggie Molecular Biology Plasma membrane protein Chromatography High Pressure Liquid Chromatography Two-dimensional gel electrophoresis medicine.diagnostic_test Chemistry Cell Membrane Protein primary structure Brain Membrane Proteins Cell Biology Peptide Fragments Membrane HPLC FT-ICR-MS |
| Zdroj: | Analytical biochemistry. 298(1) |
| ISSN: | 0003-2697 |
| Popis: | The neuronal protein reggie-2 is localized at the cytoplasmic face of the plasma membrane and participates, together with reggie-1, in the formation of plasma membrane microdomains. Reggie-2 exhibits several potential phosphorylation sites but whether the relevant sites are modified accordingly is not yet known. In order to obtain a detailed, molecular characterization of the primary structure of the native protein, an effective procedure for the isolation of the different reggie proteins from animal tissue is required. The specific properties of the proteins, particularly their membrane association and low abundance, make approaches for isolation such as affinity chromatography and 2D gel electrophoresis unfeasible. This study describes a rapid and efficient procedure for the isolation of reggie-2 by use of two consecutive HPLC steps and subsequent SDS–PAGE. The protein fractions were characterized by SDS–PAGE and Western blot analysis as well as by mass spectrometry. In the primary structure analysis by matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS), the efficiency of high-resolution Fourier-transform ion cyclotron resonance-MALDI-MS was demonstrated, enabling the direct, unequivocal, and sensitive characterization of posttranslationally and/or chemically modified proteins. |
| Databáze: | OpenAIRE |
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