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

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.