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 posttranslational 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 β-endorphin 1-31) was increased ∼7-fold in the Cpe ^fat/fat pituitary extracts. In contrast, acetyl β-endorphin 1-26 was present at ∼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.