Mass spectrometric detection of proopiomelanocortin (POMC)-related peptides following molecular cloning of POMC cDNA in bigeye tuna Thunnus obesus

Several pituitary hormones, including adrenocorticotropin (ACTH), melanotropin (MSH) and beta-endorphin, are generated from a common precursor protein, proopiomelanocortin (POMC). In fish, in addition to steroidogenesis of ACTH and melanogenesis of MSH, immunomodulating activity has been found in some POMC-related peptides. To investigate the functions of these peptides in the homologous system, it is necessary to establish a convenient detection method for the peptides. The present study aimed to establish a method for the detection of POMC-related peptides in bigeye tuna Thunnus obesus using a small amount of tissue sample, but not requiring peptide purification. We first determined the nucleotide sequence of tuna POMC cDNA. The cDNA was composed of 1084 base pairs (excluding the poly A tail) that encoded POMC consisting of 222 amino acids. We then fractionated an acid-acetone extract of one pituitary by reverse-phase high performance liquid chromatography (HPLC) and determined the molecular weight of each separated peptide by mass spectrometry. Consequently, we detected eight POMC-related peptides by comparing the values to the deduced amino acid sequence. Thus, the present study enabled the detection of POMC-related peptides from a small amount of tissue without the use of several purification steps.