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

Area of Science:

• Biochemistry
• Proteomics
• Analytical Chemistry

Background:

• Bone tissue presents challenges for proteomic analysis due to its dense matrix and high collagen content.
• Low-abundance proteins crucial for bone physiology are often overshadowed in traditional proteomic studies.
• Efficient extraction and quantification of bone proteins are vital for understanding bone diseases and developing therapies.

Purpose of the Study:

• To develop and validate a rapid, sequential two-step extraction protocol for enhanced bone proteomic analysis.
• To reduce collagen interference without enzymatic digestion.
• To enable simultaneous metabolome analysis for multiomics applications.

Main Methods:

• A novel sequential two-step extraction protocol was developed and compared against two existing methods (enzymatic digestion and four-step extraction).
• Performance was evaluated based on protein quantification reproducibility, variance, collagen content, processing time, and instrument time.
• Metabolite extraction using chloroform-methanol was integrated to assess its impact on protein quantification.

Main Results:

• The novel protocol reproducibly quantified 4,518 proteins across a 4-order dynamic range.
• It achieved comparable quantification to a four-step protocol but reduced extraction and measurement time by half.
• The protocol significantly outperformed a collagenase-based method, identifying more proteins and requiring less time.

Conclusions:

• The developed protocol enhances proteome coverage and reduces collagen interference in bone tissue analysis.
• It offers a time-efficient alternative for robust protein quantification and enables simultaneous metabolome analysis.
• This method supports comprehensive multiomics analyses for bone disease assessment and therapeutic development.