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 p lasma membrane and participates, together with reggie-1, in the formation o f plasma membrane microdomains. Reggie-2 exhibits several potential phospho rylation sites but whether the relevant sites are modified accordingly is n ot 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 assoc iation and low abundance, make approaches for isolation such as affinity ch romatography and 2D gel electrophoresis unfeasible. This study describes a rapid and efficient procedure for the isolation of reggie-2 by use of two c onsecutive HPLC steps and subsequent SDS-PAGE. The protein fractions were c haracterized by SDS-PAGE and Western blot analysis as well as by mass spect rometry. In the primary structure analysis by matrix-assisted laser desorpt ion-ionization mass spectrometry (MALDI-MS), the efficiency of high-resolut ion Fourier-transform ion cyclotron resonance-MALDI-MS was demonstrated, en abling the direct, unequivocal, and sensitive characterization of posttrans lationally and/or chemically modified proteins. (C) 2001 Academic Press.