Comprehensive evaluation of plasma biomarkers in patients with abdominal aortic aneurysm using high-throughput, label-free quantitative proteomic profiling

The use of comprehensive unbiased proteomic evaluations coupled with physiological and clinical insight offers the possibility to enhance our understanding of disease mechanisms and identify potential biomarkers for diagnosis and prognosis of cardiovascular disease. In this methods and resources article, we present the methodologies used for a workflow leveraging a high-throughput, large-scale mass spectrometry-based technology for label-free quantitative proteomic profiling coupled with physiological and clinical assessment. Using this approach, we analyzed a total of 505 plasma samples at baseline and 1- and 2-yr follow-up from patients enrolled in the Non-Invasive Treatment of Abdominal Aortic Aneurysm Clinical Trial. We successfully identified a dataset comprising over 1,000 distinct proteins. Collecting samples longitudinally provides built-in validation, allowing for direct comparisons within the same patient over time. We provide a comprehensive collection and analytical data plan tailored for such a large dataset, which encompasses data acceptance criteria, integrity checks, and database validation. We also provide methods on statistical testing, temporal cross validation, and biological and clinical validation. This resource outlines a template for similar experiments and development of a robust statistical analysis plan that will limit false positive and false negative identifications to focus on true protein changes that may serve as diagnostic or prognostic indicators.<b>NEW & NOTEWORTHY</b> We describe methods for developing a high-throughput, label-free quantitative proteomics workflow integrated with physiological and clinical assessments to investigate cardiovascular disease mechanisms and identify potential biomarkers. This resource outlines a validated, longitudinal plasma proteomics pipeline-including data quality controls, statistical testing, and biological validation-applied to over 500 samples from the N-TA³CT trial, offering a reproducible framework for future biomarker discovery studies.