Quantitation of neuropeptides in Cpe(fat)/Cpe(fat) mice using differential isotopic tags and mass spectrometry.
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.
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Fa-yun
FY
Che
Lloyd D
LD
Fricker
D000595:Amino Acid Sequence; D000818:Animals; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000172:deficiency* / Q000235:genetics; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000032:analysis*; D011499:Protein Processing, Post-Translational / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D009479:Neuropeptides; D002268:Carboxypeptidases; D043423:Carboxypeptidase H