Layer-Resolved Corneal Proteomics Enabled by Laser Ablation Sampling and LC–MS/MS

German researchers used liquid chromatography–tandem mass spectrometry (LC–MS/MS)–based differential quantitative proteomics to achieve spatially resolved profiling of the human corneal proteome, following nanosecond mid-infrared laser (NIRL) layer-by-layer ablation of consecutive corneal tissue layers and optical coherence tomography (OCT)–guided visualization of layer thickness. This approach enabled direct sampling from intact fresh-frozen tissue with three-dimensional resolution. The research team states that their method provides layer-specific insights into corneal biology and clinical processes, clarifies associated signaling pathways, and allows mapping of potential pharmacological targets. A paper based on their work was published in Investigative Ophthalmology & Visual Science (1).

In addition to its critical role in light refraction, which contributes over 70% of the eye's total refractive power, the cornea also performs other important ocular functions, such as protection against biological and chemical damage, biomechanical stability, and structural resilience (2,3). Traumatic injuries, infections, and corneal diseases (including Fuchs' endothelial dystrophy and keratoconus) are a primary cause of blindness worldwide and affect millions of individuals (4–7).Even with a variety of existing pharmacological and surgical treatments, a deeper functional understanding of corneal biology and disease mechanisms is necessary for the development of more effective therapeutic strategies (8).

Forthis study, successive layers of corneal tissue from five human corneas were ablated using a nanosecond mid-infrared laser, with ablation depth verified by optical coherence tomography. Each layer was analyzed by LC–MS/MS-based quantitative proteomics to generate a spatial map of the corneal proteome. This analysis identified 4454 proteins. In addition, a clear distinction between proteome clusters reflecting the tissue layers within the cornea was observed. A variety of biological processes were localized in the individual segments of the cornea, and an increase in metabolic proteins in the epithelium suggested high metabolic activity and regeneration processes. Notably, the stroma exhibited significantly higher abundances of immune-related proteins. A distinct proteomic profile was also discovered in the subepithelial region, which is clinically involved in corneal wound healing (1).

“Our findings, write the authors of the study (1), “offer proteomic insights into key biological processes in the cornea, including immune response, wound healing, and corneal homeostasis. Additionally, they provide a possibility for mapping the abundance of pharmacological target molecules, which could enhance the understanding of therapeutic mechanisms and drug delivery strategies.”

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References

1. Schadwinkel, H. M.; Nissen, P.; Moritz, M. et al. Layer-Specific Proteomic Profiling of the Human Cornea Reveals Insights Into Structure and Biological Function. Invest. Ophthalmol. Vis. Sci. 2026, 67 (1), 44. DOI: 10.1167/iovs.67.1.44
2. Blackburn, B. J.; Jenkins, M. W.; Rollins, A. M. et al. A Review of Structural and Biomechanical Changes in the Cornea in Aging, Disease, and Photochemical Crosslinking. Front. Bioeng. Biotechnol. 2019, 7, 66. DOI: 10.3389/fbioe.2019.00066
3. Chowdhury, D. P. H.; Shah, B. H. Basics of Anatomy and Physiology of Cornea. Acta Scientific Ophthalmol. 2021, 4, 2582–3191.
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6. Kate, A.; Basu, S. Corneal Blindness in the Developing World: The Role of Prevention Strategies. F1000Res 2024, 12, 1309. DOI: 10.12688/f1000research.141037.2
7. Ting, D. S. J.; Ho, C. S.; Deshmukh, R. et al. Infectious Keratitis: An Update on Epidemiology, Causative Microorganisms, Risk Factors, and Antimicrobial Resistance. Eye2021, 35, 1084–1101.
8. Kumar, A.; Yun, H.; Funderburgh, M. L. et al. Regenerative Therapy for the Cornea. Prog. Retin. Eye Res. 2022, 87, 101011. DOI: 10.1016/j.preteyeres.2021.101011