MCW Kassem Lab - Ocular proteins in an aging rabbit model of lensectomy

Ocular proteomic and transcriptomic changes with aging in response to lensectomy in a rabbit model of lensectomy with intraocular lens insertion
Data License: CC BY 4.0 | ProteomeXchange: PXD028205 | doi: https://doi.org/10.6069/h9dg-fe61
  • Organism: New Zealand rabbit
  • Instrument: Orbitrap Fusion Lumos
  • SpikeIn: No
  • Keywords: aqueous humor, eye, coagulation, rabbit, age
  • Lab head: Iris Kassem Submitter: Jonathon Young
Abstract
Children that undergo intraocular surgery, especially for cataracts, often have significant postoperative challenges compared to adults. The mechanisms establishing the differences in the ocular postoperative response between a child and adult are not well elucidated. Rabbits are an excellent animal model for investigating aging differences, treatment options, and surgical techniques for anterior chamber surgical interventions due to similarities in anterior chamber size and decreased postoperative response with age. Previous studies have shown that juvenile rabbits experience a dense fibrin scar following intraocular lens (IOL) insertion, and proteins related to coagulation and inflammation significantly increase in the aqueous humor (AH) following the procedure. This investigation seeks to determine how RNA transcripts and proteins change with age in a rabbit model of lensectomy with IOL insertion. Specifically, we characterize the transcription profile of the cornea and iris/ciliary body (CB) in juvenile and adult rabbits and determine how proteins related to coagulation and inflammation change with age in the AH following lensectomy with IOL insertion. In this work, juvenile and adult rabbits underwent lensectomy with IOL insertion, and AH was collected immediately prior to surgery and on post-operative day 3 (POD 3). Following a clinical exam on POD3, the cornea and iris/CB tissues were dissected and underwent RNA-extraction using a RNeasy Extraction kit. Cornea and iris/CB were also collected from rabbits that did not undergo any surgical invention. Proteins related to coagulation and inflammation were assessed in AH samples using targeted mass spectrometry via parallel reaction monitoring. While juvenile rabbits have greater fibrin formation following intraocular surgery compared to older rabbits, this change does not appear to be related to relative abundance levels of coagulation and inflammatory proteins in the AH. Transcription levels of genes from a variety of immune-response and inflammatory pathways reflected significant increases when comparing operated to unoperated ocular tissues, indicating the significant impact that surgery has on each ocular structure. This work further advances our understanding of how the rabbit eye transcriptome and proteome change in response to surgery with age as we seek to ultimately identify the mechanisms responsible for the exaggerated postoperative responses in pediatric intraocular surgery procedures.
Experiment Description
10 microliters of each aqueous humor (AH sample) was aliquoted and diluted with 100mM ammonium bicarbonate, 40% Invitrosol, 20% acetonitrile (MeCN), and 5.52 mM tris(2-carboxyethyl)phosphine (TCEP) to bring the samples to a final concentration of 5mM TCEP for reduction of the proteins. Samples were then incubated for 30 minutes at 1400 rpm at 25 °C in a thermomixer (Eppendorf, Hamburg, Germany). 100 mM iodoacetamide was added to bring samples to 10mM for protein alkylation and then mixed in the dark for 30 minutes at 1400rpm at 37 °C in a thermomixer. Pierce Trypsin/Lys-C (Thermo Scientific, Waltham, MA) mix was added in a 1:20 trypsin-to-protein ratio for overnight incubation (~18 hours) at 37 °C 1400 rpm in the thermomixer. Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles (hydrophylic and hydrophobic; GE Life Sciences, Sheffield, UK) were prepared at a 1:1 ratio to create a particle suspension, and peptides were prepared for proteomic analysis using the SP2 method as previously described (1). Briefly, 8 microliters of particle solution was added to the samples to create a 20:1 particle to peptide ratio, and 100% MeCN was added to bring the final concentration of MeCN to 95%. Samples were mixed by pipetting up and down 3-5x to make sure the particles were well dispersed, allowed to settle for two minutes, and placed on a magnetic rack. The supernatant was removed, and samples were washed with 100% MeCN to cover the particles completely. Samples were returned to the magnetic rack and allowed to sit for 60 seconds. Next, another 100% MeCN wash was performed, and the supernatant discarded. The particles were then reconstituted in a solution consisting of 2% MeCN and water, vortexed, and allowed to settle for one minute. The supernatant (now containing the peptides) was collected and transferred to a new microfuge tube. Samples were then acidified with 10% formic acid for a final concentration of 0.1% acid. The sample was then spun at 14000 rpm for 10 minutes to precipitate any remaining particles and the supernatant was collected. Peptides were quantified using a Pierce Quantitative Fluorometric Peptide assay, and each sample was diluted to 25ng/microliter in 2% MeCN with 0.1% formic acid. Peptide Retention Time Calibration (PRTC) Mixture (Thermo Fisher Scientific, Waltham, MA) was added to each sample at a final concentration of 4nM to enable retention time calibration and assessment of instrument performance throughout acquisition. Equal volumes of each sample were combined into a single “pooled quality-control (QC)” mixture. Based upon a previous study (2), a selected list of 31 proteins of interest related to coagulation and complement cascades were analyzed by MS. Targeted quantitation by parallel reaction monitoring (PRM) was performed using a Dionex UltiMate 3000 RSLCnano system in-line with an Orbitrap Fusion Lumos Tribrid mass spectrometer. Samples (as two replicate injections) were queued in a randomized order. Each peptide was scheduled with a set 10-minute scheduled retention time window, and resulting MS data were processed using Skyline. Retention times and ions were manually aligned and selected. (1) Waas M, Pereckas M, Jones Lipinski RA, Ashwood C, Gundry RL. SP2: Rapid and Automatable Contaminant Removal from Peptide Samples for Proteomic Analyses. J Proteome Res. 2019;18(4):1644-56. PMC6571012 (2) Young JB, Keppel TR, Waas M, Salmon AE, Buchberger AR, Skumatz CMB, Gundry RL, Kassem IS. Quantitative proteomic analysis of aqueous humor after rabbit lensectomy reveals differences in coagulation and immunomodulatory proteins. Mol Omics. 2020;16(2):126-37. PMID:32031200
Sample Description
Aqueous humor (AH) is a clear fluid in the front part of the eye. We collected this fluid from juvenile and adult rabbits before surgery, and three days following surgery. We sought to determine how proteins in the AH related to coagulation and inflammation change with age following surgical intervention.
Created on 9/3/21, 6:47 PM
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Aging_Run_1_Analysis_Use_no_precursor_32247_Removed_2021-06-14_15-09-39.sky.zip2021-09-03 18:47:003110310347155