Cathepsin K; Cleavage sites identification; Degradomics; High resolution mass spectrometry; Nano liquid chromatography; Type I collagen digestion; Cell Biology; Clinical Biochemistry; Biochemistry; General Medicine; Analytical Chemistry
Abstract :
[en] Since the late 1990s, cathepsin K cleavage sites in type I collagen have been extensively studied due to its ability to release bone resorption biomarkers such as CTX and NTX. However, gel-based methods and N-sequencing used in these studies lack sensitivity, especially for small to medium peptides. In this work, we propose a degradomics mass spectrometry-based workflow that combines protein digestion, Nano-LC-UDMSE, and several software tools to identify cathepsin K cleavage sites. This workflow not only identified previously known cleavage sites, but also discovered new ones. Multiple cleavage hotspots were found and described in type I α1 and type I α2 collagen, many of which coincided with pyridinoline crosslinks, known to stabilize the triple helix. Our results allowed us to establish a chronology of digestion and conclude that cathepsin K preferentially cleaves the extremities of type I collagen before the helical part. We also found that cathepsin K preferentially cleaves amino acid residues with long and hydrophobic lateral chains at the beginning of digestion, whereas no preferred amino acid residues were identified later in the digestion. In conclusion, our workflow successfully identified new cleavage sites and can be easily applied to other proteins or proteases.
Disciplines :
Laboratory medicine & medical technology
Author, co-author :
Demeuse, Justine ; Université de Liège - ULiège > Département de pharmacie > Chimie médicale
Massonnet, Philippe ; Centre Hospitalier Universitaire de Liège - CHU > > Service de chimie clinique
Schoumacher, Matthieu ; Université de Liège - ULiège > Unités de recherche interfacultaires > Centre Interdisciplinaire de Recherche sur le Médicament (CIRM)
Grifnée, Elodie ; Centre Hospitalier Universitaire de Liège - CHU > > Service de chimie clinique
Huyghebaert, Loreen ; Centre Hospitalier Universitaire de Liège - CHU > > Service de chimie clinique
Dubrowski, Thomas ; Centre Hospitalier Universitaire de Liège - CHU > > Service de chimie clinique
Peeters, Stéphanie ; Centre Hospitalier Universitaire de Liège - CHU > > Service de chimie clinique
Le Goff, Caroline ; Centre Hospitalier Universitaire de Liège - CHU > > Service de chimie clinique
Cavalier, Etienne ; Centre Hospitalier Universitaire de Liège - CHU > > Service de chimie clinique
Language :
English
Title :
Innovative workflow for the identification of cathepsin K cleavage sites in type I collagen.
Publication date :
25 August 2023
Journal title :
Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
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