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    Identification of proteins affected by increased intraocular pressure in the glaucomatous female mouse retina by label-free proteomics
    (2023) Zaman, Khadiza; Morgan, Autumn B.; Nguyen, Vien; Prokai-Tatrai, Katalin; Inman, Denise; Prokai, Laszlo
    Purpose: Mass spectrometry-based retina proteomics using animal models of human diseases has enabled novel insights into ocular neuropathology’s such as in glaucoma, as it holds promise for disease biomarker discovery. However, publicly accessible data on retina proteins affected by ocular hypertension (OHT) in animal models utilized males, or sex was not disclosed. Recently, female animals were chosen to advance therapeutic antibody development against glaucomatous neurodegeneration with retina proteomics support. Therefore, our retinal proteomics-based investigation intended to fill a knowledge gap by focusing on OHT-induced changes of protein expressions in the glaucomatous female retinae compared to normotensive controls. Methods: Proteins were extracted from the retinae of normotensive female mice (control, n=5) and OHT mice (n=5) in which increase of intraocular pressure was induced by the magnetic microbead method. After reduction, alkylation and digestion by trypsin, bottom-up shotgun proteomics analyses of the samples were done using data-dependent nanoflow liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) on a hybrid Orbitrap instrument (Thermo Fisher Scientific). MS/MS spectra were searched against the UniProt mouse protein sequence database using the SEQUEST search engine in Proteome Discoverer (version 2.4; Thermo Fisher Scientific). Validation of proteins identifications using stringent criteria and label-free quantifications (LFQ) employing spectral counting to detect regulated proteins between groups using t-tests were performed using Scaffold (version 5.1.2; Proteome Software). Targeted proteomics on selected biomarkers was designed and analyzed using SkylineTM (MacCoss Lab software). Mapping to protein interaction networks and biological processes was done through Ingenuity Pathway Analysis® (IPA®, Qiagen). Results: Our discovery driven data-dependent nanoflow LC–ESI-­MS/MS analyses covered nearly 1200 retinal proteins with <1% false discovery rate. Among these proteins, 168 were significantly affected by OHT based on LFQ. Bioinformatics analyses by IPA® revealed important diseases and functions triggered by OHT pertaining to neurological and ophthalmic pathologies. The topmost protein interaction network represented neurological disease, organismal injury and abnormalities. The molecule activity predictor of IPA® revealed important canonical pathways, including inhibition of synaptogenesis signaling and mitochondrial dysfunction leading to degeneration of central nervous system tissue. Another prominent protein interaction network represented nervous system development and function, as well as organ development. In addition, this network also displayed downregulation of neuroprotective crystallins owing to OHT. Neuronal crystallins have been identified not only as biomarkers to monitor the progression of OHT-induced retinal neuropathy and evaluate neuroprotective interventions, but also as potential druggable targets or possible protein therapeutics to prevent glaucomatous neurodegeneration. Parallel reaction monitoring-based targeted proteomics validation of significant OHT-regulated retina proteins are currently underway to establish them as potential preclinical biomarkers and/or therapeutic targets. In addition, our studies will be expanded to investigate sex as a biological variable affecting ocular neurodegeneration associated with glaucoma. Conclusion: We anticipate that biological information one can derive from our dataset at the protein expression level will provide inspiration for future hypothesis-driven experimental studies focusing on knowledge gaps involving the biology of glaucomatous neurodegeneration.
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    (2023) Kapic, Ammar; Zaman, Khadiza; Nguyen, Vien; Prokai-Tatrai, Katalin; Prokai, Laszlo
    Purpose: Androgen deprivation therapy (ADT) remains the primary treatment strategy for inhibiting prostate cancer progression. However, systemic ablation of androgen-mediated signaling induces various metabolic disorders, cognitive decline, and osteoporosis. Therefore, like in menopausal women, 17-β-estradiol (E2) supplementation has been suggested as a treatment to reduce side effects associated with ADT. A recent clinical trial utilizing transdermal estrogen patches reported reduced osteoporosis markers and hot flashes. Estrogen receptors ER) are expressed in the male reproductive system and play a role alongside androgens in maintaining function and growth. Under normal physiological conditions, increased E2 concentrations induce an inhibitory effect on the size of the male reproductive organs, including the seminal vesicles (SV); however, under androgen depletion, E2 supplementation has been reported to reduce the atrophy of the SV in mice. In this study, we report for the first time a discovery-driven proteomic analysis of E2’s effects on the SV in mice under the conditions of surgical castration to model patients undergoing ADT. Methods: Surgically castrated mice (n=4) were subcutaneously injected with E2 (treated group) or vehicle (control) daily for five days and sacrificed to obtain SV. Proteins were extracted, reduced, alkylated, and digested with trypsin for analyses using data-dependent microflow liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) on LTQ Orbitrap Velos ProTM (Thermo Fisher Scientific). MS/MS data was searched against the UniProt Mouse protein database using Sequest in Proteome Discoverer (Thermo Fisher Scientific) and MaxQuant (Max Planck Institute). Validation of protein and label-free quantification (LFQ), combining spectral counting and total TIC, were performed using Scaffold (Proteome Software) to identify significantly affected proteins. Post-hoc t-test was performed to identify differences in protein abundances between groups. Regulated proteins we mapped to protein interaction networks and biological functions employing Ingenuity Pathway Analysis® (IPA®, Qiagen). Targeted proteomics- sed validation of significant candidate proteins is ongoing, and data will be analyzed using Skyline TM (MacCoss Lab, University of Washington). Results: Our discovery-driven LC–ESI-MS/MS analyses identified 7000 proteins with high confidence from the SV of E2-treated and control mice. IPA®-based bioinformatics of the E2-regulated proteins showed molecular and cellular functions-associated enrichment of carbohydrate metabolism, DNA replication, recombination, and repair, as well as free radical scavenging. The topmost regulated protein interaction network represented cell cycle, cell signaling, and small molecule biochemistry. Enhanced activation of the estrogen receptor β (ESR2) was implicated by the molecule activity predictor (MAP) tool of IPA®. Additionally, MAP predicted that the protein interaction represented within this network might impact disease and physiological processes associated with the proliferation of prostate cancer and regulation of gonadal cells. Furthermore, we were able to screen several preclinical biomarkers that participate in androgen receptor activity, modulating ER-mediated transcription and reproductive system development and function. Targeted proteomics-based validation of these biomarkers is ongoing. Conclusion: Our study aims to provide an in-depth account of the alterations occurring at the protein level in the SVs in response to E2 supplementation during ADT and to select and validate preclinical biomarkers for prognostic and therapeutic applications.