Quantitation of Neuropeptides in Cpefat/Cpefat Mice Using Differential Isotopic Tags and Mass Spectrometry

Abstract

Neuroendocrine peptides play important roles as intercellular messengers. We previously developed a technique to isolate and identify a large number of neuroendocrine peptides from Cpe^fat/Cpe^fat mice (Che, F.; et al. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9971−6); these mice lack carboxypeptidase E activity and this defect causes an accumulation of neuropeptide intermediates that contain C-terminal Lys or Arg residues (Naggert, J. K.; et al. Nat. Genet. 1995, 10, 135−42). In the present study, we have developed a differential isotopic-labeling technique that can be used to quantitate changes in neuropeptide levels in Cpe^fat/Cpe^fat mouse tissues. Samples are treated with either the H₆ or the D₆ form of acetic anhydride, peptides that contain C-terminal basic amino acids are isolated by affinity chromatography on anhydrotrypsin agarose, and the isolated peptides are analyzed by mass spectrometry. Measurement of the regulation of pituitary peptides in response to dehydration showed a decrease in vasopressin. The general method described in this report should be widely applicable to a large number of neuroendocrine peptides, known and novel, in a variety of regulatory paradigms.