Analysis of the proteolysis of bioactive peptides using a peptidomics approach.
Identifying the peptidases that inactivate bioactive peptides (e.g., peptide hormones and neuropeptides) in mammals is an important unmet challenge. This protocol describes a recent approach that uses liquid chromatography-mass spectrometry (LC-MS) peptidomics to identify endogenous cleavage sites of a bioactive peptide; it also addresses the subsequent biochemical purification of a candidate peptidase on the basis of these cleavage sites and the validation of the candidate peptidase's role in the physiological regulation of the bioactive peptide by examining a peptidase-knockout mouse. We highlight the successful application of this protocol in the discovery that insulin-degrading enzyme (IDE) regulates physiological calcitonin gene-related peptide (CGRP) levels, and we detail the key stages and steps in this approach. This protocol requires 7 d of work; however, the total time for this protocol is highly variable because of its dependence on the availability of biological reagents such as purified enzymes and knockout mice. The protocol is valuable because it expedites the characterization of mammalian peptidases, such as IDE, which in certain instances can be used to develop novel therapeutics.
Department of Chemical Engineering, Soongsil University, Seoul, South Korea.
Yun-Gon
YG
Kim
Anna Mari
AM
Lone
Alan
A
Saghatelian
D000818:Animals; D015740:Calcitonin Gene-Related Peptide / Q000378:metabolism; D002853:Chromatography, Liquid; D055786:Gene Knockout Techniques; D007339:Insulysin / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000378:metabolism; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D010447:Peptide Hydrolases; D007339:Insulysin; D015740:Calcitonin Gene-Related Peptide