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Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels
Published on: February 22, 2019
Quantitative proteomic analysis of aqueous humor after rabbit lensectomy reveals differences in coagulation and
Jonathon B Young1, Theodore R Keppel, Matthew Waas
1Cell Biology, Neurobiology, & Anatomy, Medical College of Wisconsin, The Eye Institute, 925 N. 87th Street, Milwaukee, WI 53226, USA. ikassem@mcw.edu.
Insights
Pediatric intraocular surgery shows increased scarring in children. This study identifies key proteins in the aqueous humor of juvenile rabbits, revealing changes in complement, coagulation, and immunosuppression proteins post-surgery.
Area of Science:
- Ophthalmology
- Proteomics
- Pediatric Surgery
Background:
- Children exhibit heightened scarring and inflammation after intraocular surgery compared to adults.
- The precise reasons for this exaggerated immune response in pediatric patients remain unclear.
- Proteins are critical mediators in wound healing and postoperative scarring.
Purpose of the Study:
- To identify and quantify proteins linked to the heightened postoperative immune response in pediatric intraocular surgery.
- To investigate the aqueous humor proteome changes in a juvenile rabbit model following lensectomy with intraocular lens insertion.
Main Methods:
- Quantitative proteomics, including label-free quantitation (LFQ) via data-dependent and data-independent acquisition.
- Targeted quantitation using parallel reaction monitoring (PRM) for specific proteins.
- Surgical procedures in juvenile rabbits included paracentesis, corneal incision, lensectomy, and lensectomy with IOL insertion.
Main Results:
- Total protein concentration in aqueous humor increased with surgical complexity.
- Proteins involved in complement and coagulation cascades showed increased abundance post-surgery.
- Proteins associated with ocular immunosuppression demonstrated decreased abundance after surgical procedures.
Conclusions:
- This study provides the first step-wise proteomic view of the aqueous humor response to intraocular surgery in a juvenile model.
- Findings highlight alterations in specific protein pathways, offering insights into pediatric postoperative responses.
- This research lays the groundwork for future therapeutic strategies targeting proteins to mitigate complications in pediatric intraocular surgery.
Abstract:
Compared to adults, children experience increased postoperative scarring and inflammation following intraocular surgery. While the underlying causes of the exaggerated immune response in children are not understood, proteins play key roles in postoperative scarring and wound healing processes. To identify and quantify proteins associated with the robust postoperative immune response, this study applied quantitative proteomics approaches to a juvenile rabbit model of lensectomy with intraocular lens (IOL) insertion. 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 was collected immediately prior and three days after each procedure. Semi-quantitative protein discovery was achieved by label-free quantitation using data dependent and data independent acquisition modes. Based on the discovery results, targeted quantitation by parallel reaction monitoring of 3 proteins of interest, fibrinogen-beta chain, transforming growth factor beta-2, and retinol binding protein 3, was used to confirm the observed quantitative trends. Total protein concentration levels increased with each progressive surgical step of lensectomy with IOL insertion. Proteins related to the complement and coagulation cascades were found to increase in relative abundance, while proteins related to ocular immunosuppression decreased in abundance following surgery. These data provide insights into the postoperative response by providing the first surgical step-wise views of the AH proteome before and after surgery. Overall, this work provides the foundation for future investigations targeting specific proteins for therapeutic interventions aimed at minimizing postoperative complications after pediatric intraocular surgery.
