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Effective protein extraction combined with data independent acquisition analysis reveals a comprehensive and quantifiable insight into the proteomes of articular cartilage and subchondral bone
Osteoarthritis and Cartilage ( IF 7 ) Pub Date : 2021-09-20 , DOI: 10.1016/j.joca.2021.09.006
L Bundgaard 1 , E Åhrman 2 , J Malmström 2 , U Auf dem Keller 3 , M Walters 4 , S Jacobsen 4
Affiliation  

Objective

The objectives of this study was to establish a sensitive and reproducible method to map the cartilage and subchondral bone proteomes in quantitative terms, and mine the proteomes for proteins of particular interest in the pathogenesis of osteoarthritis (OA). The horse was used as a model animal.

Design

Protein was extracted from articular cartilage and subchondral bone samples from three horses in triplicate by pressure cycling technology or ultrasonication. Digested proteins were analysed by data independent acquisition based mass spectrometry. Data was processed using a pre-established spectral library as reference database (FDR 1%).

Results

We identified to our knowledge the hitherto most comprehensive quantitative cartilage (1758 proteins) and subchondral bone (1482 proteins) proteomes in all species presented to date. Both extraction methods were sensitive and reproducible and the high consistency of the identified proteomes (>97% overlap) indicated that both methods preserved the diversity among the extracted proteins. Proteome mining revealed a substantial number of quantifiable cartilage and bone matrix proteins and proteins involved in osteogenesis and bone remodeling, including ACAN, BGN, PRELP, FMOD, COMP, ACP5, BMP3, BMP6, BGLAP, TGFB1, IGF1, ALP, MMP3, and collagens. A number of proteins, including COMP and TNN, were identified in different protein isoforms with potential unique biological roles.

Conclusion

We have successfully developed two sensitive and reproducible non-species specific workflows enabling a comprehensive quantitative insight into the proteomes of cartilage and subchondral bone. This facilitates the prospect of investigating the molecular events at the osteochondral unit in the pathogenesis of OA in future projects.



中文翻译:

有效的蛋白质提取与数据独立采集分析相结合,揭示了对关节软骨和软骨下骨蛋白质组的全面和可量化的见解

客观的

本研究的目的是建立一种灵敏且可重复的方法,以定量方式绘制软骨和软骨下骨蛋白质组图,并挖掘蛋白质组以寻找在骨关节炎 (OA) 发病机制中特别感兴趣的蛋白质。这匹马被用作模型动物。

设计

通过压力循环技术或超声处理从三匹马的关节软骨和软骨下骨样品中提取蛋白质,一式三份。通过基于数据独立采集的质谱分析消化的蛋白质。使用预先建立的光谱库作为参考数据库 (FDR 1%) 处理数据。

结果

据我们所知,我们鉴定了迄今为止所有物种中迄今为止最全面的定量软骨(1758 种蛋白质)和软骨下骨(1482 种蛋白质)蛋白质组。两种提取方法都是灵敏且可重复的,并且鉴定的蛋白质组的高度一致性(> 97%重叠)表明两种方法都保留了提取蛋白质之间的多样性。蛋白质组挖掘揭示了大量可量化的软骨和骨基质蛋白和参与成骨和骨重塑的蛋白质,包括 ACAN、BGN、PRELP、FMOD、COMP、ACP5、BMP3、BMP6、BGLAP、TGFB1、IGF1、ALP、MMP3 和胶原蛋白。许多蛋白质,包括 COMP 和 TNN,被鉴定为具有潜在独特生物学作用的不同蛋白质亚型。

结论

我们成功开发了两种敏感且可重复的非物种特异性工作流程,能够对软骨和软骨下骨的蛋白质组进行全面的定量分析。这有助于在未来的项目中研究 OA 发病机制中骨软骨单元的分子事件的前景。

更新日期:2021-09-20
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