THE PHOSPHORYLATION SITE AND DESMETHIONYL N-TERMINUS OF DROSOPHILA PHOSRESTIN-I IN-VIVO DETERMINED BY MASS-SPECTROMETRIC ANALYSIS OF PROTEINS SEPARATED BY 2-DIMENSIONAL GEL-ELECTROPHORESIS

Post-translational modifications of proteins play crucial roles in mod ulating many cellular processes, In order to understand the physiologi cal roles of post-translational protein modifications it is imperative to determine the nature of the change in chemical structure involved in each protein modification, In our earlier work, we developed a meth od for the study of protein modification through a streamlined combina tion of two-dimensional gel electrophoresis, in-gel digestion, high pe rformance liquid chromatography and electrospray ionization tandem qua drupole mass spectrometry, Using this method we determined the in vivo phosphorylation site of phosrestin I to be Ser(366). Since our earlie r work described the method only briefly, we will present a full descr iption of the method in this paper, In addition, by using this method, we also show that the N-terminus of phosrestin I is desmethionylated in vivo, These techniques are easily adapted to the study of other pro teins and will serve as powerful tools for the study of post-translati onal protein modifications in general.