Systematic Comparison of Bone Proteome Extraction Methods to Allow for Integrated Proteomics–Metabolomics Correlation

Bone tissue poses significant challenges for proteomic analysis due to its dense, mineral-rich matrix and predominance of collagen, overshadowing low-abundance proteins critical for understanding bone physiology during LC-MS/MS-based proteomic analysis. In this study, we present a rapid sequential two-step extraction protocol designed to enhance proteome coverage, reduce collagen interference without using collagenase, and ensure robust quantification while enabling simultaneous metabolome analysis. We systematically compared it with two previously reported methods, which attempt to reduce collagen content through enzymatic collagen digestion or by employing four sequential extractions. Performance was evaluated based on reproducible protein quantification, variance, collagen content, processing, and instrument time. Our protocol reproducibly quantified 4,518 proteins across a dynamic range of 4 orders of magnitude. It demonstrated only marginally inferior quantification performance compared to the four-step protocol while reducing extraction and measurement time by half. Further, it significantly outperformed the collagenase-based method, which quantified only 2,689 proteins. Incorporating a chloroform-methanol metabolite extraction only led to a minimal reduction in quantifiable proteins, making the protocol suitable for multiomics applications. In conclusion, this protocol facilitates comprehensive coverage of proteins after metabolite extraction, enabling comprehensive multiomics analyses and aiding in the assessment of bone diseases and therapeutic developments.