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

Area of Science:

• Proteomics
• Ophthalmology
• Molecular Biology

Background:

• The retinal pigment epithelium/choroid (RC) is crucial for retinal health but challenging to study due to limited sample sizes.
• Understanding RC dysfunction is vital for treating sight-threatening diseases like neovascular age-related macular degeneration (nvAMD).

Purpose of the Study:

• To optimize liquid chromatography-field asymmetric ion mobility spectrometry-mass spectrometry (LC-FAIMS-MS/MS) for in-depth proteomic analysis of RC tissue.
• To identify proteins and pathways involved in nvAMD pathogenesis using an optimized workflow.

Main Methods:

• Utilized an optimized LC-FAIMS-MS/MS workflow with an Orbitrap Fusion Lumos mass spectrometer.
• Analyzed RC tissue from a mouse model of nvAMD.
• Integrated multiomics data for differential expression and pathway analysis.

Main Results:

• Identified 7047 proteins in RC tissue, a significant increase over conventional LC-MS/MS (5500 proteins).
• Revealed 295 significantly altered proteins in nvAMD, including ECM remodeling (HTRA1, CCDC80) and immune response (SYK, CTSS) regulators.
• Highlighted pathways in neutrophil chemotaxis, ECM organization, and PI3K-Akt signaling, suggesting immune-ECM crosstalk in choroidal neovascularization (CNV).

Conclusions:

• The optimized LC-FAIMS-MS/MS technique enhances proteomic depth for RC tissue analysis.
• This approach provides novel insights into nvAMD molecular mechanisms.
• Identified potential therapeutic targets for nvAMD and related conditions.