Plenary Poster 33rd ASM of the Australian & New Zealand Bone & Mineral Society 2023

Chemical digestion-assisted extracellular matrix profiling of differentiating osteoblasts (#103)

Lay Thant 1 2 3 , Masaru Kaku 4 , Hlaing Pwint Phyu 4 , Azusa Dobashi 4 , Yoshito Kakihara 2 , Isao Saito 1 , Katsumi Uoshima 4
  1. Division of Orthodontics, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
  2. Division of Dental Pharmacology, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
  3. Center of Advanced Oral Science, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
  4. Division of Bio-prosthodontics, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan

Introduction: Quantification of extracellular matrix (ECM) proteins is challenging due to their insoluble nature, however, chemical digestion using Hydroxylamine (HA) has recently been introduced to overcome this problem. We evaluated the efficacy of HA for ECM solubilization in comparison to traditional chaotropic buffer and successfully established a chemical digestion-assisted protocol that provides a more in-depth ECM coverage. During osteoblastic differentiation, the proportion of the ECM is rich in proteoglycans and glycoproteins, and they were gradually replaced by fibrillar collagens, which then mineralized to form bone. Although the transcriptional regulation of osteoblasts is relatively well understood, the changes in the ECM during osteoblastic differentiation remain elusive.

Objective: The objective of this study was to present the compositional changes in the ECM proteome of differentiating osteoblasts using chemical digestion-assisted proteomics.

Methods: Mouse MC3T3-E1 cells were cultured in a differentiation medium to obtain osteoblasts-derived ECM. The cells were harvested by RIPA buffer after 1, 2, 3, and 4 weeks, and the ECM fraction of the cells was obtained by centrifugation. We employed the established chemical digestion-assisted protocol, which features serial protein extraction with Guanidine hydrochloride (Gnd-HCl) and Gnd-HCl+HA. Tryptic peptides were prepared from the entire protein extracts and analyzed by mass spectrometry. The obtained proteome list was curated with the Matrisome database.

Results: In the differentiating osteoblasts, the proportion of collagen in the matrisome increased each week from 71.4% to 87.6%, with the increasing type I collagen proportion from ~60% to 80% among collagens. The proportions of proteoglycans and glycoproteins were highest at 1 week of differentiation with 4.9% and 8.7%, respectively, and decreased thereafter.

Conclusion: Using chemical digestion-assisted proteomics, we present the changes in the ECM profile of differentiating osteoblasts, which partly illustrate the ECM maturation process involved in bone formation.