Nanoparticle-enabled plasma proteomics of a mouse atherosclerosis model.

Background: Dyslipidemia, marked by elevated LDL-cholesterol (LDL-C), is a major risk factor for coronary heart disease. Mouse models, such as Ldlr-/- mice that develop atherosclerosis and metabolic disorders when fed a high-fat diet (HFD), are indispensable for studying disease mechanisms and identifying potential biomarkers.

Objectives: We aimed to profile the plasma proteins of a widely studied experimental atherosclerosis model with a primary goal to detect low-abundant proteins.

Methods: Ldlr-/- mice were fed a chow diet or HFD or 3 (n = 27 per diet) or 6 months (n = 12 per diet). Plasma samples were processed using nanoparticle technology (Proteograph® XT Assay; Seer, Inc), and peptides were analyzed using the Orbitrap Astral (Thermo Fisher Scientific) in data-independent acquisition mode. For tissue proteomics, ten aortas were pooled for n = 4 pools per diet and month, and n = 6 livers per diet and month. Peptides were analyzed on the Orbitrap Exploris 480 in data-dependent mode. Proteomes were queried against the Tabula Muris mouse single-cell, STRING, and Gene Ontology databases, and queried against a genome-wide association list of 419 risk loci for coronary artery disease.

Results: We sequenced 5,080 plasma proteins, surpassing previous reports by 10-fold. The prototypical apolipoproteins and complement factors were the most intense proteins, whereas proteins associated with cytokine/chemokine signaling represent the previously uncharted mouse plasma proteome. We divided the proteome into quartiles (Q1-Q4) to monitor sweeping changes over time. Proteins with a sustained enrichment in HFD (n = 705) are indicative of liver cell subtypes (Tabula Muris). Whereas proteins that moved up from the lower quartiles - Q2 (n = 228), Q3 (115) and Q4 (63) - indicate leukocytes and fibroblasts, and endothelial cells; demonstrating that signatures of inflammation and endothelial activation increase with disease progression. Notably, 86 and 146 proteins were increased at 3 and 6 months, including MMP-12 and COL6A3. Classical apolipoproteins exhibited heterogeneous responses - SAA3 and APOC2 increased, while APOA1, APOE, and LCAT decreased with high-fat feeding, indicating impaired high-density lipoprotein (HDL) functionality. Proteins shared between plasma and aorta were enriched for extracellular matrix components, while those overlapping with liver reflected metabolic processes. Finally, 120 CAD-associated proteins from human GWAS were detected in Ldlr-/- plasma, of which 4, including lipoprotein lipase, exhibited an increase in abundance with HFD.

Conclusions: Nanoparticle-dependent proteome enrichment coupled to mass spectrometry may allow us to identify novel plasma biomarkers in Ldlr-/- mice and facilitate monitoring of candidate proteins associated with human disease mechanisms in preclinical interventional studies, thereby opening new avenues for understanding disease pathology and uncovering understudied molecular contributors.