Affinity purification of neuropeptide precursors from mice lacking carboxypeptidase E activity

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Citation (Scopus)

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages199-208
Number of pages10
DOIs
StatePublished - Jan 1 2018

Publication series

NameMethods in Molecular Biology
Volume1719
ISSN (Print)1064-3745

Fingerprint

Carboxypeptidase H
Neuropeptides
Peptides
Peptide Hormones
Neuroendocrine Cells
Secretory Pathway
Affinity Chromatography
Sepharose

Keywords

  • Affinity chromatography
  • Anhydrotrypsin
  • Carboxypeptidase E
  • Neuropeptide

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Fricker, L. D. (2018). Affinity purification of neuropeptide precursors from mice lacking carboxypeptidase E activity. In Methods in Molecular Biology (pp. 199-208). (Methods in Molecular Biology; Vol. 1719). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-7537-2_13

Affinity purification of neuropeptide precursors from mice lacking carboxypeptidase E activity. / Fricker, Lloyd D.

Methods in Molecular Biology. Humana Press Inc., 2018. p. 199-208 (Methods in Molecular Biology; Vol. 1719).

Research output: Chapter in Book/Report/Conference proceedingChapter

Fricker, LD 2018, Affinity purification of neuropeptide precursors from mice lacking carboxypeptidase E activity. in Methods in Molecular Biology. Methods in Molecular Biology, vol. 1719, Humana Press Inc., pp. 199-208. https://doi.org/10.1007/978-1-4939-7537-2_13
Fricker LD. Affinity purification of neuropeptide precursors from mice lacking carboxypeptidase E activity. In Methods in Molecular Biology. Humana Press Inc. 2018. p. 199-208. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-7537-2_13
Fricker, Lloyd D. / Affinity purification of neuropeptide precursors from mice lacking carboxypeptidase E activity. Methods in Molecular Biology. Humana Press Inc., 2018. pp. 199-208 (Methods in Molecular Biology).
@inbook{748ecf948a6f46c6ad3ad1097ba493f9,
title = "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.",
keywords = "Affinity chromatography, Anhydrotrypsin, Carboxypeptidase E, Neuropeptide",
author = "Fricker, {Lloyd D.}",
year = "2018",
month = "1",
day = "1",
doi = "10.1007/978-1-4939-7537-2_13",
language = "English (US)",
series = "Methods in Molecular Biology",
publisher = "Humana Press Inc.",
pages = "199--208",
booktitle = "Methods in Molecular Biology",

}

TY - CHAP

T1 - Affinity purification of neuropeptide precursors from mice lacking carboxypeptidase E activity

AU - Fricker, Lloyd D.

PY - 2018/1/1

Y1 - 2018/1/1

N2 - 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.

AB - 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.

KW - Affinity chromatography

KW - Anhydrotrypsin

KW - Carboxypeptidase E

KW - Neuropeptide

UR - http://www.scopus.com/inward/record.url?scp=85042559291&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85042559291&partnerID=8YFLogxK

U2 - 10.1007/978-1-4939-7537-2_13

DO - 10.1007/978-1-4939-7537-2_13

M3 - Chapter

T3 - Methods in Molecular Biology

SP - 199

EP - 208

BT - Methods in Molecular Biology

PB - Humana Press Inc.

ER -