Relative quantitation of peptides in wild-type and Cpefat/fat mouse pituitary using stable isotopic tags and mass spectrometry

✍ Scribed by Fa-Yun Che; Reeta Biswas; Lloyd D. Fricker

• Publisher: John Wiley and Sons
• Year: 2005
• Tongue: English
• Weight: 228 KB
• Volume: 40
• Category: Article
• ISSN: 1076-5174
• DOI: 10.1002/jms.742

✦ Synopsis

Abstract

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 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. Copyright © 2005 John Wiley & Sons, Ltd.