Characterization of bioactive peptides in bovine adrenal medulla by a combination of fast HPLC and ESI-MS

A method based upon a combination of fast high-performance liquid chromatography (HPLC) and electrospray ionization (ESI)–mass spectrometry (MS) is developed for the analysis of bioactive peptides in bovine adrenal medulla. The fast HPLC uses a short column (33 mm×4.6 mm) packed with nonporous silica-based C-18 stationary phase. Prior to HPLC separation, the medulla was homogenized and the peptide-rich fraction was isolated from it by solid-phase extraction. In-source collision-induced dissociation and tandem MS were used to obtain the sequence of the suspected peptides. Several peptides, including Met–Enk, Leu–Enk, Leu–Enk–Lys, bovine adrenal medullary (BAM)-12 (Met–Enk–RRVGRPE), Leu–Enk–Arg, and YGGT, were unambiguously identified. The first four peptides are the products of proenkephalin A precursor protein and Leu–Enk–Arg belongs to the dynorphin family and is derived from proenkephalin B (prodynorphin) precursor. The database search revealed that YGGT is a part of the sequence of five different precursor proteins.