Dataset Information

Combined retinal proteome datasets in response to atropine treatment using iTRAQ and SWATH-MS based proteomics approaches in guinea pig myopia model.

ABSTRACT: Atropine, a non-selective muscarinic antagonist, is known to slow down myopia progression in human adolescents and in several animal models. However, its underlying molecular mechanism is unclear. The present work built a monocular form-deprivation myopia (FDM) guinea pig model, using facemasks as well as atropine treatment on FDM eyes for 2 and 4 weeks. Retinal protein changes in response to the FDM and effects of topical administration of atropine were screened for the two periods using fractionated isobaric tags for a relative and absolute quantification (iTRAQ) approach coupled with nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) (n=24, 48 eyes). Retinal tissues from another cohort receiving 4-weeks FDM with atropine treatment (n=12, 24 eyes) with more significant changes were subjected to sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics for further protein target confirmation. A total of 1695 proteins (8875 peptides) and 5961 proteins (51871 peptides) were identified using iTRAQ and SWATH approaches, respectively. Using the Paragon algorithm in the ProteinPilot^TM software, the three most significantly up-regulated and down-regulated proteins that were commonly found in both ITRAQ and SWATH experiments are presented. All raw data generated from the work were submitted and published in the Peptide Atlas public repository (http://www.peptideatlas.org/) for general release (Data ID PASS01507).

SUBMITTER: Zhu Y

PROVIDER: S-EPMC7708793 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Combined retinal proteome datasets in response to atropine treatment using iTRAQ and SWATH-MS based proteomics approaches in guinea pig myopia model.

Zhu Ying Y Bian Jingfang J Lu Daqian D Wang Qiong Q Gong Boteng B Li King-Kit KK Yu Fengjuan F Cheung Jimmy Ka-Wai JK Ji Xiaowen X Zhang Hongmei H Du Bei B Nian Hong H To Chi-Ho CH Wei Ruihua R Lam Thomas Chuen TC

Data in brief 20201117

Atropine, a non-selective muscarinic antagonist, is known to slow down myopia progression in human adolescents and in several animal models. However, its underlying molecular mechanism is unclear. The present work built a monocular form-deprivation myopia (FDM) guinea pig model, using facemasks as well as atropine treatment on FDM eyes for 2 and 4 weeks. Retinal protein changes in response to the FDM and effects of topical administration of atropine were screened for the two periods using fracti ...[more]

PMID: 33304948

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Project description:Purpose: Atropine, a non-selective muscarinic antagonist, effectively slows down myopia progression in human adolescents and several animal models. However, the underlying molecular mechanism is unclear. The current study investigated retinal protein changes of form-deprived myopic (FDM) guinea pigs in response to topical administration of 1% atropine gel (10 g/L). Methods: At the first stage, the differentially expressed proteins were screened using fractionated isobaric tags for a relative and absolute quantification (iTRAQ) approach, coupled with nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) (n = 24, 48 eyes) using a sample pooling technique. At the second stage, retinal tissues from another cohort with the same treatment (n = 12, 24 eyes) with significant ocular changes were subjected to label-free sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics for orthogonal protein target confirmation. The localization of Alpha-synuclein was verified using immunohistochemistry and confocal imaging. Results: A total of 1,695 proteins (8,875 peptides) were identified with 479 regulated proteins (FC ≥ 1.5 or ≤0.67) found from FDM eyes and atropine-treated eyes receiving 4-weeks drug treatment using iTRAQ-MS proteomics. Combining the iTRAQ-MS and SWATH-MS datasets, a total of 29 confident proteins at 1% FDR were consistently quantified and matched, comprising 12 up-regulated and 17 down-regulated proteins which differed between FDM eyes and atropine treated eyes (iTRAQ: FC ≥ 1.5 or ≤0.67, SWATH: FC ≥ 1.4 or ≤0.71, p-value of ≤0.05). Bioinformatics analysis using IPA and STRING databases of these commonly regulated proteins revealed the involvement of the three commonly significant pathways: EIF2 signaling; glycolysis; and dopamine secretion. Additionally, the most significantly regulated proteins were closely connected to Alpha-synuclein (SNCA). Using immunostaining (n = 3), SNCA was further confirmed in the inner margin of the inner nuclear layer (INL) and spread throughout the inner plexiform layer (IPL) of the retina of guinea pigs. Conclusion: The molecular evidence using next-generation proteomics (NGP) revealed that retinal EIF2 signaling, glycolysis, and dopamine secretion through SNCA are implicated in atropine treatment of myopia in the FDM-induced guinea pig model.

Project description:Most of the previous myopic animal studies employed a single-candidate approach and lower resolution proteomics approaches that were difficult to detect minor changes, and generated limited systems-wide biological information. Hence, a complete picture of molecular events in the retina involving myopic development is lacking. Here, to investigate comprehensive retinal protein alternations and underlying molecular events in the early myopic stage, we performed a data-independent Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) based proteomic analysis coupled with different bioinformatics tools in pigmented guinea pigs after 4-day lens-induced myopia (LIM). Myopic eyes compared to untreated contralateral control eyes caused significant changes in refractive error and choroid thickness (p < 0.05, n = 5). Relative elongation of axial length and the vitreous chamber depth were also observed. Using pooled samples from all individuals (n = 10) to build a species-specific retinal ion library for SWATH analysis, 3202 non-redundant proteins (with 24,616 peptides) were identified at 1% global FDR. For quantitative analysis, the 10 individual retinal samples (5 pairs) were analyzed using a high resolution Triple-TOF 6600 mass spectrometry (MS) with technical replicates. In total, 37 up-regulated and 21 down-regulated proteins were found significantly changed after LIM treatment (log2 ratio (T/C) > 0.26 or < -0.26; p ≤ 0.05). Data are accepted via ProteomeXchange with identifier PXD025003. Through Ingenuity Pathways Analysis (IPA), "lipid metabolism" was found as the top function associated with the differentially expressed proteins. Based on the protein abundance and peptide sequences, expression patterns of two regulated proteins (SLC6A6 and PTGES2) identified in this pathway were further successfully validated with high confidence (p < 0.05) using a novel Multiple Reaction Monitoring (MRM) assay on a QTRAP 6500+ MS. In summary, through an integrated discovery and targeted proteomic approach, this study serves as the first report to detect and confirm novel retinal protein changes and significant biological functions in the early LIM mammalian guinea pigs. The study provides new workflow and insights for further research to myopia control.

Project description:Atropine, a muscarinic antagonist, is known to inhibit myopia progression in several animal models and humans. However, the mode of action is not established yet. In this study, we compared quantitative iTRAQ proteomic analysis in the retinas collected from control and lens-induced myopic (LIM) mouse eyes treated with atropine. The myopic group received a (-15D) spectacle lens over the right eye on postnatal day 10 with or without atropine eye drops starting on postnatal day 24. Axial length was measured by optical low coherence interferometry (OLCI), AC-Master, and refraction was measured by automated infrared photorefractor at postnatal 24, 38, and 52 days. Retinal tissue samples were pooled from six eyes for each group. The experiments were repeated twice, and technical replicates were also performed for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. MetaCore was used to perform protein profiling for pathway analysis. We identified a total of 3882 unique proteins with <1% FDR by analyzing the samples in replicates for two independent experiments. This is the largest number of mouse retina proteome reported to date. Thirty proteins were found to be up-regulated (ratio for myopia/control > global mean ratio + 1 standard deviation), and 28 proteins were down-regulated (ratio for myopia/control < global mean ratio - 1 standard deviation) in myopic eyes as compared with control retinas. Pathway analysis using MetaCore revealed regulation of ?-aminobutyric acid (GABA) levels in the myopic eyes. Detailed analysis of the quantitative proteomics data showed that the levels of GABA transporter 1 (GAT-1) were elevated in myopic retina and significantly reduced after atropine treatment. These results were further validated with immunohistochemistry and Western blot analysis. In conclusion, this study provides a comprehensive quantitative proteomic analysis of atropine-treated mouse retina and suggests the involvement of GABAergic signaling in the antimyopic effects of atropine in mouse eyes. The GABAergic transmission in the neural retina plays a pivotal role in the maintenance of axial eye growth in mammals.

Dataset Information

Combined retinal proteome datasets in response to atropine treatment using iTRAQ and SWATH-MS based proteomics approaches in guinea pig myopia model.

Publications

Combined retinal proteome datasets in response to atropine treatment using iTRAQ and SWATH-MS based proteomics approaches in guinea pig myopia model.

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