Relative quantitation of peptides in wild-type and Cpe(fat/fat) mouse pituitary using stable isotopic tags and mass spectrometry.
Cpe(fat/fat) mice have a point mutation in the coding region of the carboxypeptidase E gene that renders the enzyme inactive. As a result, these mice have reduced levels of several neuropeptides and greatly increased levels of the peptide processing intermediates that contain C-terminal basic residues. However, previous studies examined a relatively small number of neuropeptides. In the present study, we used a quantitative peptidomics approach with stable isotopic labels to examine the levels of pituitary peptides in Cpe(fat/fat) mice relative to wild-type mice. Pituitary extracts from mutant and wild type mice were labeled with the stable isotopic label [3-(2,5-dioxopyrrolidin-1-yloxycarbonyl)propyl]trimethylammonium chloride containing nine atoms of hydrogen or deuterium. Then, the two samples were pooled and analyzed by liquid chromatography/mass spectrometry (LC/MS). The relative abundance of peptides was determined from a comparison of the intensities of the heavy and light peaks. Altogether, 72 peptides were detected in the Cpe(fat/fat) and/or wild-type mouse pituitary extracts of which 53 were identified by MS/MS sequencing. Several peptides identified in this analysis represent previously undescribed post-translational processing products of known pituitary prohormones. Of the 72 peptides detected in pituitary, 17 were detected only in the Cpe(fat/fat) mouse extracts; these represent peptide processing intermediates containing C-terminal basic residues. The peptides common to both Cpe(fat/fat) and wild-type mice were generally present at 2-5-fold lower levels in the Cpe(fat/fat) mouse pituitary extracts, although some peptides were present at equal levels and one peptide (acetyl beta-endorphin 1-31) was increased approximately 7-fold in the Cpe(fat/fat) pituitary extracts. In contrast, acetyl beta-endorphin 1-26 was present at approximately 10-fold lower levels in the Cpe(fat/fat) pituitary, compared with wild-type mice. The finding that many peptides are substantially decreased in Cpe(fat/fat) pituitary is consistent with the broad role for carboxypeptidase E in the biosynthesis of numerous neuropeptides.
Che
Fa-Yun
FY
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Biswas
Reeta
R
Fricker
Lloyd D
LD
D000595:Amino Acid Sequence; D000818:Animals; D043423:Carboxypeptidase H / Q000172:deficiency / Q000502:physiology*; D002268:Carboxypeptidases / Q000502:physiology; D002853:Chromatography, Liquid; D003903:Deuterium; D013058:Mass Spectrometry; D051379:Mice; D008820:Mice, Obese; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000032:analysis* / Q000737:chemistry; D010902:Pituitary Gland / Q000737:chemistry*; D011333:Pro-Opiomelanocortin / Q000032:analysis / Q000737:chemistry; D014020:Tissue Extracts / Q000737:chemistry
D009479:Neuropeptides; D014020:Tissue Extracts; D011333:Pro-Opiomelanocortin; D003903:Deuterium; D002268:Carboxypeptidases; C078874:carboxypeptidase D; D043423:Carboxypeptidase H