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Quantitative proteomic analysis of aqueous humor in a juvenile rabbit lensectomy model reveals surgery stage specific differences in coagulation, complement, and ocular immunosuppressive proteins

ABSTRACT: Purpose: To identify and quantify the proteins associated with the robust postoperative immune response in a juvenile rabbit model of lensectomy with intraocular lens (IOL) insertion. Methods: Twenty-six 6-7 week-old New Zealand White rabbits underwent unilateral portions of lensectomy with IOL insertion including: anterior chamber paracentesis, corneal incision with wound suture, lensectomy only, and lensectomy with IOL insertion. Aqueous humor (AH) was collected immediately prior and three days after each procedure. Semi-quantitative discovery of proteins was achieved using liquid chromatography tandem mass spectrometry (LC-MS/MS). Based on discovery results, targeted quantitation utilizing stable isotopically labeled (SIL) peptides for 3 proteins of interest, fibrinogen-beta chain, transforming growth factor beta-2, and retinol binding protein 3, were used to confirm the observed quantitative trends.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Oryctolagus Cuniculus (ncbitaxon:9986)

SUBMITTER: Iris Kassem

PROVIDER: MSV000084150 | MassIVE | Mon Jul 29 08:38:00 BST 2019

REPOSITORIES: MassIVE

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Project description:Introduction: The optical elements of the eye – cornea, lens, and vitreous humor – are avascular tissues, and their nutrition and waste removal are provided by aqueous humor (AH). The AH production occurs through the active secretion and the passive diffusion/ultrafiltration of blood plasma. The comparison of the metabolomic profiles of aqueous humor (AH) and plasma is important for understanding of the mechanisms of biochemical processes and metabolite transport taking place in vivo in ocular tissues. Objectives: The work is aimed at the determination of concentrations of a wide range of most abundant metabolites in the human AH, the comparison of the metabolomic profiles of AH and serum, and the analysis of the post-mortem metabolomic changes in these two biological fluids. Methods: The quantitative metabolomic profiling was carried out with the use of two independent methods – high-frequency 1H NMR spectroscopy and HPLC with high-resolution ESI-MS detection (LC-MS). Results: The concentrations of 71 most abundant metabolites in blood serum and AH from living patients and human cadavers have been measured. It has been found that the level of ascorbate in AH is by two orders of magnitude higher than that in serum; the levels of other metabolites are either similar to that in serum, or differ from that by a factor of 2-5. The post-mortem metabolomic composition of both serum and AH undergoes rapid and strong changes. Conclusion: The differences between the metabolomic profiles of AH and serum for majority of metabolites can be attributed to the metabolic activity of the ocular tissues leading to the lack or excess of some metabolites, while the high concentration of ascorbate in AH demonstrates the activity of ascorbate-specific pumps at the blood-aqueous border. The post-mortem metabolomic changes are caused by the disruption of the major biochemical cycles and cell lysis. These changes should be taken into account in the analysis of disease-induced changes in post-mortem samples of the ocular tissues.

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Quantitative proteomic analysis of aqueous humor in a juvenile rabbit lensectomy model reveals surgery stage specific differences in coagulation, complement, and ocular immunosuppressive proteins

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