Significance: The cornea is essential for focusing light and protecting the eye. Corneal diseases are a leading cause of blindness worldwide and remain challenging to treat effectively. Conditions such as severe keratoconus often require corneal transplantation. Understanding the layer-specific functions of the cornea is crucial for improving therapy, yet most studies lack spatial resolution. This highlights the need for high-resolution sampling techniques for detailed proteomic analysis of corneal layers.
Methods: We utilized nanosecond infrared laser (NIRL) ablation setup (Navolić and Moritz et al., Anal Chem, 2023) to sample 24 consecutive layers from the central and paracentral regions of fresh-frozen corneal tissue from five patients. Ablation depth was monitored using optical coherence tomography (OCT). Proteins were extracted through cold evaporation, collected on a glass slide for tryptic digestion, and analysed via LC-MS/MS on an Orbitrap Exploris 480 mass spectrometer. The proteomic profiles of all layers from each patient were analyzed.
Results: We quantified 4,454 proteins, with 3,615 found in at least three samples. Significant protein abundance differences were observed across layers, with endothelium layers showing distinct separation from others. Layers from each patient were annotated to four main clusters aligned with the ablation order. Known pathways associated with corneal tissue layers could be assigned to the clusters. Novel findings included immune response activities localized primarily in the stromal region. While core proteomic signatures were consistent across patients, substantial inter-patient variability individualized considerations in therapeutic applications.
Conclusion: NIRL-based layer-wise sampling enables detailed proteomic analysis of the human cornea, providing detailed insights into the layer-specific biological pathways. This method offers a foundation for advancing targeted therapeutic approaches for corneal diseases.
Acknowledgement: This work was supported by a DFG grant (SCHL 406/21-1) in the funding program: “New instruments for research”.