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. SUMMARY: Abdominal aortic aneurysm (AAA) is a life-threatening, non-communicable disease that remains underdiagnosed and poorly understood within the medical community. Furthermore, there is a lack of an effective medical therapy, aside from surgical intervention, that compels clinicians to address general cardiovascular risk factors for disease management. Thus, fishing putative new biomarkers and therapeutic targets from aneurysmatic tissue using untargeted proteomic approaches has emerged as a relevant strategy for the development of tools for earlier diagnosis, effective disease management, and a deeper understanding of the pathophysiology of AAA. However, given the heterogeneity of AAA tissue, the reproducibility of the results may be partially affected by the absence of a standardized method for protein extraction while ensuring high efficiency in protein yield. Therefore, this study aimed to address a key bottleneck in proteomic analysis of aneurysmatic study-heterogeneity in sample processing. Herein, we report the optimization of a protocol for AAA tissue homogenization and maximize protein extraction. A standard operating procedure (SOP) to process AAA tissue toward downstream proteomics applications is shared to enable more reliable and comparable data across studies and ultimately bolster the translation of tissue proteomics into clinically relevant tools for vascular medicine.