Isoaspartate-containing galanin in rat hypothalamus

The isoaspartate residue is a spontaneous, time-dependent post-translational modification (PTM) of proteins and peptides, associated with in vivo protein aggregation and changes in molecule lifetime. While this is considered a slow modification impacting long lived proteins, surprisingly, we observed this PTM at high levels within the relatively short-lived neuropeptide galanin (Gal). The combination of liquid chromatography-trapped ion mobility mass spectrometry and protein Isoaspartyl methyltransferase assays demonstrated that 20 ± 2% of the mature Gal contain L-Isoaspartate residue in the hypothalamus of Rattus norvegicus. Aspartate in Gal isomerizes spontaneously under mildly acidic conditions within 48 h in vitro, much faster than previously assumed. Gal with the L-isoaspartate PTM significantly enhanced fibril formation. Transmission electron microscopy revealed differences in morphology of fibrils formed by D17Isoasparte Gal compared to the unmodified peptide. Observed characteristics of D17Isoasparte Gal suggest a potential mechanism for the development of in vivo Gal fibril deposits previously reported in the brain.