The Phosphorylation Site and Desmethionyl N-Terminus of Drosophila Phosrestin I in vivo Determined by Mass Spectrometric Analysis of Proteins Separated by Two-Dimensional Gel Electrophoresis

Abstract

Post-translational modifications of proteins play crucial roles in modulating many cellular processes. In order to understand the physiological 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 method for the study of protein modification through a streamlined combination of two-dimensional gel electrophoresis, in-gel digestion, high performance liquid chromatography and electrospray ionization tandem quadrupole mass spectrometry. Using this method we determined the in vivo phosphorylation site of phosrestin I to be Ser₃₆₆. Since our earlier work described the method only briefly, we will present a full description 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 proteins and will serve as powerful tools for the study of post-translational protein modifications in general.