Isolation and mass spectrometry based hydroxyproline mapping of type II collagen derived from Capra hircus ear cartilage

Priti Prasanna Maity,Debabrata Dutta,Sayan Ganguly,Amit Kumar Das,Kausik Kapat,Ramapati Samanta,Santanu Dhara,Krishna Dixit,Narayan Chandra Das,Amit Roy Chowdhury,Pallab Datta

doi:10.1038/s42003-019-0394-6

Abstract

Collagen II (COLII), the most abundant protein in vertebrates, helps maintain the structural and functional integrity of cartilage. Delivery of COLII from animal sources could improve cartilage regeneration therapies. Here we show that COLII can be purified from the Capra ear cartilage, a commonly available bio-waste product, with a high yield. MALDI-MS/MS analysis evidenced post-translational modifications of the signature triplet, Glycine-Proline-Hydroxyproline (G-P-Hyp), in alpha chain of isolated COLII (COLIIA1). Additionally, thirty-two peptides containing 59 Hyp residues and a few G-X-Y triplets with positional alterations of Hyp in COLIIA1 are also identified. Furthermore, we show that an injectable hydrogel formulation containing the isolated COLII facilitates chondrogenic differentiation towards cartilage regeneration. These findings show that COLII can be isolated from Capra ear cartilage and that positional alteration of Hyp in its structural motif, as detected by newly developed mass spectrometric method, might be an early marker of cartilage disorder.

Highlights

Collagen II (COLII), the most abundant protein in vertebrates, helps maintain the structural and functional integrity of cartilage
The isolated COLII is transformed into hydrogel along with Pluronic F127 in combination with Capra adipose tissue-derived stem cells (ADMSCs) towards differentiation of chondrogenic lineage in vitro
Isolation of COLII was optimized using 0.1% (w/v) pepsin digestion followed by 1.2 M NaCl precipitation to produce yield of ∼55% (Supplementary Fig. 1)

Summary

Introduction

Collagen II (COLII), the most abundant protein in vertebrates, helps maintain the structural and functional integrity of cartilage. We show that an injectable hydrogel formulation containing the isolated COLII facilitates chondrogenic differentiation towards cartilage regeneration. These findings show that COLII can be isolated from Capra ear cartilage and that positional alteration of Hyp in its structural motif, as detected by newly developed mass spectrometric method, might be an early marker of cartilage disorder. Hyp mapping is performed to validate its sequential alteration in Gly–X–Y structural motif in COLIIA1. This identification may contribute to the prediction of dysfunctional collagen leading to arthritis as well as molecular identification of COLII from other sources. The isolated COLII is transformed into hydrogel along with Pluronic F127 in combination with Capra adipose tissue-derived stem cells (ADMSCs) towards differentiation of chondrogenic lineage in vitro

Methods

Results

Conclusion