A Standard Operating Procedure for Protein Extraction From Abdominal Aortic Aneurysm Tissue: Enhancing Proteomics Applications

Purpose: The identification of putative biomarkers for AAA can be achieved through shotgun proteomics. However, tissue heterogeneity hampers its reproducible homogenization and protein extraction. Thus, we aimed to optimize a protocol to maximize protein yield and develop an SOP to foster reproducibility and accelerate translation of proteomics findings.

Experimental Design: Using a bead-beating homogenization method, we compared the effect of beads’ size, extraction cycles, beads-to-tissue mass ratio, lysis buffer volume, and chemistry on protein yield and/or qualitative and quantitative parameters of proteomics analysis (identifications, sequence coverage, coefficient of variation, functional enrichment analysis).

Results: Optimal conditions for protein extraction were achieved using 1.4 mm beads in two homogenization cycles, with a bead-to-tissue mass ratio of 30:1 and 20 µL of lysis buffer per mg of tissue. As for the buffer chemistry, RIPA is recommended to attain greater sequence coverage, while HEPES and Urea/thiourea are preferred when quantification performance is a priority. The SOP was applied to characterize the AAA tissue proteome, and key AAA pathogenesis-related pathways were highlighted by bioinformatic analysis.

Conclusions and Clinical Relevance: The SOP is well-suited for identifying and quantifying aneurysmatic tissue proteins and can be applied to accelerate the translation of putative biomarkers into clinical diagnostic/prognostic tools.