Novel approaches for the measurement of unusual post-translational modifications in neuropeptides.

摘要

Chemical modifications of the side chains of amino acid residues in peptides following translation increase the diversity of products possible from the genetic code, as well as confer appropriate biological activity upon the peptide. However, these modifications, which are crucial to the function of the peptide, can be difficult to pinpoint. In addition to determining the chemical identity of modified group, the specific amino acid residue involved must also be identified. In peptides where multiple types of modifications can alter any number of amino acid residues, this can be enormously complicated task.; Mass spectrometry (MS) has gained popularity in the analysis of post-translational modifications (PTMs), as a rapid and easily automated alternative to chromatographic techniques such as two-dimensional gel electrophoresis. Many PTMs demonstrate a characteristic mass shift associated with the chemical group that allows for easy identification using MS. PTMs can be further localized to particular amino acid residues through the application of fragmentation methods such as in-source or post-source decay.; Not all PTMs are amenable to analysis by MS. The lability of certain post-translational modifications limits the utility of MS, as the modified group can be lost in the ionization process. MALDI-MS of nitrated peptides results in photoreduction of the nitro group, yielding the sequential loss of oxygen. This PTM, however, can be stabilized by the appropriate choice of matrix, boosting the signal of the nitrated peak.; Other PTMs, such as the transformation of L-amino acid residues to the D-configuration, do not result in a measurable mass change. Capillary electrophoresis combined with mass spectrometry is used to identify cells containing a D-amino acid-containing peptide found in Aplysia. These cells are then utilized in the development of a method combining the specificity of an enzyme with liquid chromatography/mass spectrometry to locate novel D-amino acid-containing peptides in the central nervous system of Aplysia californica.