| The phosphorylation of serine, threonine and tyrosine residues plays a vital role in many aspects of protein expression and function. Protein phosphorylation is a reversible process that acts as a molecular switch, controlling pathways in metabolism, signal transduction and cell division. Because the phosphorylation of many proteins has been shown to alter the structure and function of the protein, localization of the site of modification has increasingly been studied in the last few years.; The use of many standard analytical and biochemical techniques, such as gel electrophoresis and Edman degradation, allow us to identify unmodified peptides and proteins. However, the use of these techniques to determine the sites of post-translational modification has proven to be more challenging. Because of this, mass spectrometry has been used extensively in the identification and subsequent localization of the sites of phosphorylation for many peptides and proteins. The use of electron capture dissociation (ECD) Fourier transform mass spectrometry (FTMS) has proven to be a valuable tool in the localization of many classes of modifications. Unlike other mass spectrometric dissociation techniques, ECD uses low energy electrons for the fragmentation process. In this process, the fragmentation occurs prior to energy randomization, allowing the modifications to remain intact.; In our study, an electron capture dissociation source was installed on an existing FTMS system. Both an electron filament and a dispenser cathode were used for the electron capture dissociation experiments. Once the operating parameters were optimized, several modified and unmodified peptides and small proteins were fragmented. The fragmentation efficiencies of both ECD sources were also compared. In addition, a series of phosphorylated test peptides (MARCKS-related peptides) was studied using ECD-FTMS. Resulting fragmentation patterns were used to determine a series of fragmentation rules for the dissociation of both singly and doubly phosphorylated peptides. The relative abundance of each phosphorylated and non-phosphorylated serine residue was also explored. The established rules were then used in an attempt to determine the site of phosphorylation for a synthetic peptide with a sequence identical to the phosphorylation site domain of the MARCKS protein. |
