Proteomic Signatures of Choroidal Neovascularization via Integrated LC-FAIMS-MS/MS Workflow.

Abstract

The multicellular retinal pigment epithelium/choroid (RC) tissue is pivotal in maintaining retinal homeostasis and is closely associated with sight-threatening eye diseases. However, the limited sample amount, particularly in mice, poses a great challenge in comprehensively characterizing the functional proteins of the RC in disease models. This study utilized a state-of-the-art FAIMS device coupled with an Orbitrap Fusion Lumos mass spectrometer to systematically optimize the LC, FAIMS, and MS/MS acquisition parameters for in-depth proteomic analysis of the difficultly obtained RC samples. In a mouse model of neovascular age-related macular degeneration (nvAMD), the optimized workflow effectively increased the coverage of the proteome, which enabled the identification of 7047 proteins, compared to 5500 identified by conventional LC-MS/MS. Combined with multiomics data sets across species, differential expression analysis revealed 295 significantly altered proteins in the nvAMD model, including key regulators of extracellular matrix (ECM) remodeling (HTRA1, CCDC80) and immune response (SYK, CTSS). Functional enrichment and protein-protein interaction (PPI) network analysis highlighted critical pathways involved in neutrophil chemotaxis, ECM organization, and PI3K-Akt signaling, uncovering potential crosstalk between immune dysregulation and ECM degradation in choroidal neovascularization (CNV) progression. In conclusion, the optimized LC-FAIMS-MS/MS technique presented in this study offers an enhanced depth of proteomic analysis for the RC tissue, revealing novel insights into the molecular mechanisms of nvAMD and identifying new potential therapeutic targets.