Affinity purification of neuropeptide precursors from mice lacking carboxypeptidase E activity

Abstract

Peptidomic techniques are powerful tools to identify peptides in a biological sample. This protocol describes a targeted peptidomic approach that uses affinity chromatography to purify peptides that are substrates of carboxypeptidase E (CPE), an enzyme present in the secretory pathway of neuroendocrine cells. Many CPE products function as neuropeptides and/or peptide hormones, and therefore represent an important subset of the peptidome. Because CPE removes C-terminal Lys and Arg residues from peptide-processing intermediates, organisms lacking CPE show a large decrease in the levels of the mature forms of most neuropeptides and peptide hormones, and a very large increase in the levels of the processing intermediates that contain C-terminal Lys and/or Arg (i.e., the CPE substrates). These CPE substrates can be purified on an anhydrotrypsin-agarose affinity resin, which specifically binds peptides with C-terminal basic residues. Not all peptides with basic C-terminal residues within a cell are CPE substrates, and these other peptides will also be purified on the anhydrotrypsin affinity column. However, a comparison of peptides purified from wild-type mice and from mice lacking CPE allows for the rapid identification of CPE substrates based on their large increase in the absence of CPE.