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.
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.
Bauer
S H
SH
Department of Biology, University of Konstanz, Konstanz, 78457, Germany.
Wiechers
M F
MF
Bruns
K
K
Przybylski
M
M
Stuermer
C A
CA
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D029941:Fish Proteins; D006054:Goldfish; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000032:analysis*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
D000906:Antibodies; D029941:Fish Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; C104436:reggie-2 protein, Carassius auratus