SWATH Based Quantitative Proteomics Reveals Significant Lipid Metabolism in Early Myopic Guinea Pig Retina
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 myo...
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| Format: | Article |
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MDPI AG
2021-04-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/9/4721 |
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| author | Jingfang Bian Ying-Hon Sze Dennis Yan-Yin Tse Chi-Ho To Sally A. McFadden Carly Siu-Yin Lam King-Kit Li Thomas Chuen Lam |
| author_facet | Jingfang Bian Ying-Hon Sze Dennis Yan-Yin Tse Chi-Ho To Sally A. McFadden Carly Siu-Yin Lam King-Kit Li Thomas Chuen Lam |
| author_sort | Jingfang Bian |
| collection | DOAJ |
| 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 (<i>p</i> < 0.05, <i>n</i> = 5). Relative elongation of axial length and the vitreous chamber depth were also observed. Using pooled samples from all individuals (<i>n</i> = 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; <i>p</i> ≤ 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 (<i>SLC6A6</i> and <i>PTGES2</i>) identified in this pathway were further successfully validated with high confidence (<i>p</i> < 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. |
| first_indexed | 2024-03-10T11:50:02Z |
| format | Article |
| id | doaj.art-436bdd043359472b9d19d100fb50bf42 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T11:50:02Z |
| publishDate | 2021-04-01 |
| publisher | MDPI AG |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-436bdd043359472b9d19d100fb50bf422023-11-21T17:46:55ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-04-01229472110.3390/ijms22094721SWATH Based Quantitative Proteomics Reveals Significant Lipid Metabolism in Early Myopic Guinea Pig RetinaJingfang Bian0Ying-Hon Sze1Dennis Yan-Yin Tse2Chi-Ho To3Sally A. McFadden4Carly Siu-Yin Lam5King-Kit Li6Thomas Chuen Lam7Centre for Myopia Research, School of Optometry, The Hong Kong Polytechnic University, Kowloon, Hong Kong, ChinaCentre for Myopia Research, School of Optometry, The Hong Kong Polytechnic University, Kowloon, Hong Kong, ChinaCentre for Myopia Research, School of Optometry, The Hong Kong Polytechnic University, Kowloon, Hong Kong, ChinaCentre for Myopia Research, School of Optometry, The Hong Kong Polytechnic University, Kowloon, Hong Kong, ChinaSchool of Psychology, College of Engineering, Science and the Environment, University of Newcastle, Callaghan, NSW 2308, AustraliaCentre for Myopia Research, School of Optometry, The Hong Kong Polytechnic University, Kowloon, Hong Kong, ChinaCentre for Myopia Research, School of Optometry, The Hong Kong Polytechnic University, Kowloon, Hong Kong, ChinaCentre for Myopia Research, School of Optometry, The Hong Kong Polytechnic University, Kowloon, Hong Kong, ChinaMost 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 (<i>p</i> < 0.05, <i>n</i> = 5). Relative elongation of axial length and the vitreous chamber depth were also observed. Using pooled samples from all individuals (<i>n</i> = 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; <i>p</i> ≤ 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 (<i>SLC6A6</i> and <i>PTGES2</i>) identified in this pathway were further successfully validated with high confidence (<i>p</i> < 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.https://www.mdpi.com/1422-0067/22/9/4721SWATH-MSproteomicsmyopiaretinaguinea pigslipid metabolism |
| spellingShingle | Jingfang Bian Ying-Hon Sze Dennis Yan-Yin Tse Chi-Ho To Sally A. McFadden Carly Siu-Yin Lam King-Kit Li Thomas Chuen Lam SWATH Based Quantitative Proteomics Reveals Significant Lipid Metabolism in Early Myopic Guinea Pig Retina International Journal of Molecular Sciences SWATH-MS proteomics myopia retina guinea pigs lipid metabolism |
| title | SWATH Based Quantitative Proteomics Reveals Significant Lipid Metabolism in Early Myopic Guinea Pig Retina |
| title_full | SWATH Based Quantitative Proteomics Reveals Significant Lipid Metabolism in Early Myopic Guinea Pig Retina |
| title_fullStr | SWATH Based Quantitative Proteomics Reveals Significant Lipid Metabolism in Early Myopic Guinea Pig Retina |
| title_full_unstemmed | SWATH Based Quantitative Proteomics Reveals Significant Lipid Metabolism in Early Myopic Guinea Pig Retina |
| title_short | SWATH Based Quantitative Proteomics Reveals Significant Lipid Metabolism in Early Myopic Guinea Pig Retina |
| title_sort | swath based quantitative proteomics reveals significant lipid metabolism in early myopic guinea pig retina |
| topic | SWATH-MS proteomics myopia retina guinea pigs lipid metabolism |
| url | https://www.mdpi.com/1422-0067/22/9/4721 |
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