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pmid:34445296
Differential Retinal Protein Expression in Primary and Secondary Retinal Ganglion Cell Degeneration Identified by Integrated SWATH and Target-Based Proteomics.
Jacky M K Kwong · Joseph Caprioli · Ying H Sze · Feng J Yu · King K Li · Chi H To · Thomas C Lam
International journal of molecular sciences · 2021 doi:10.3390/ijms22168592
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Complete file
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Unilateral partial optic nerve transection (pONT) was performed on adult Wistar rats and their retinas were harvested 2 weeks later. To confirm the separation of primary and secondary RGC degeneration, immunohistochemistry of RNA binding protein with multiple splicing (RBPMS) and glial fibrillary acidic protein (GFAP) was performed on retinal whole-mounts. Retinal proteomes in the temporal and nasal quadrants were evaluated with high resolution hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS), and SWATH-based acquisition, and their expression was compared to the corresponding retinal quadrant in contralateral control eyes and further validated by multiple reaction monitoring mass spectrometry (MRM-MS). A total of 3641 proteins (FDR < 1%) were identified using QTOF-MS. The raw data are available via ProteomeXchange with the identifier PXD026783. Bioinformatics data analysis showed that there were 37 upregulated and 25 downregulated proteins in the temporal quadrant, whereas 20 and five proteins were upregulated and downregulated, respectively, in the nasal quadrant, respectively (n = 4, p < 0.05; fold change ≥ 1.4-fold or ≤0.7). Six proteins were regulated in both the temporal and the nasal quadrants, including CLU, GFAP, GNG5, IRF2BPL, L1CAM, and CPLX1. Linear regression analysis indicated a strong association between the data obtained by means of SWATH-MS and MRM-MS (temporal, R2 = 0.97; nasal, R2 = 0.96). Gene ontology analysis revealed statistically significant changes in the biological processes and cellular components of primary RGC degeneration. The majority of the significant changes in structural, signaling, and cell death proteins were associated with the loss of RGCs in the area of primary RGC degeneration. The combined use of SWATH-MS and MRM-MS methods detects and quantifies regional changes of retinal protein expressions after localized injury. Future investigation with this integrated approach will significantly increase the understanding of diverse processes of progressive RGC degeneration from a proteomic prospective.", "claims": [{"id": "claim-method", "statement": "Samples were normalized by most likely ratio (MLR) normalization with a paired t-test used to detect significantly differentiated proteins in biological samples among experimental groups.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:34445296", "locator": "sections[9] \"4.5. 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Results", "row_count": 4, "column_count": 7}, "table": {"label": "Table 1", "caption": "Protein and peptide identification in retinal samples 2 weeks after pONT.", "rows": [["", "Sample Number", "Pooled Sample *", "MarkerView **"], ["", "1", "2", "3", "4"], ["Protein", "3560", "3270", "3504", "3587", "3641", "3173"], ["Peptide", "24,310", "20,800", "23,569", "25,080", "25,313", "18,336"]], "row_count": 4, "column_count": 7, "numeric_cells": 16, "header_rows": 2, "locator": "section[2]/table-wrap[1]#ijms-22-08592-t001"}}, {"id": "asset:table:pmid:34445296:2", "role": "table", "format": "jats-table", "media_type": "application/vnd.jats.table+json", "uri": null, "sha256": "sha256:83c8967f52b93500929893ac5f48679510f5453f298094999865b08b1a811504", "bytes": 6832, "access": "open", "state": "verified", "reader": "read_table", "reader_status": "available", "identifier": "section[2]/table-wrap[2]#ijms-22-08592-t002", "version": null, "note": "Protein regulation in the temporal quadrant 2 weeks after pONT compared to contralateral control (p < 0.05 and fold-change log2 > 0.43 (upregulation) or ≤ −0.43 (downregulation)). R = right; L = left; FC = fold change.", "structure": {"pmcid": "PMC8395271", "section_index": 2, "section_title": "2. Results", "row_count": 65, "column_count": 5}, "table": {"label": "Table 2", "caption": "Protein regulation in the temporal quadrant 2 weeks after pONT compared to contralateral control (p < 0.05 and fold-change log2 > 0.43 (upregulation) or ≤ −0.43 (downregulation)). 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(Hu-antigen D) (HuD) (Paraneoplastic encephalomyelitis antigen HuD)", "0.69", "−0.53", "0.00"], ["P97846", "Contactin-associated protein 1 (Caspr) (Caspr1) (Neurexin IV) (Neurexin-4) (Paranodin) (p190)", "0.69", "−0.53", "0.02"], ["Q6PW52", "GABA-A gamma2 long isoform (Gamma-aminobutyric acid A receptor, gamma 2) (Gamma-aminobutyric acid receptor subunit gamma-2)", "0.68", "−0.55", "0.01"], ["O35760", "Isopentenyl-diphosphate Delta-isomerase 1 (EC 5.3.3.2) (Isopentenyl pyrophosphate isomerase 1) (IPP isomerase 1) (IPPI1)", "0.68", "−0.56", "0.01"], ["G3V679", "Transferrin receptor protein 1 (Transferrin receptor, isoform CRA_a)", "0.68", "−0.56", "0.02"], ["P12839", "Neurofilament medium polypeptide (NF-M) (160 kDa neurofilament protein) (Neurofilament 3) (Neurofilament triplet M protein)", "0.66", "−0.60", "0.04"], ["P62762", "Visinin-like protein 1 (VILIP) (21 kDa CABP) (Neural visinin-like protein 1) (NVL-1) (NVP-1)", "0.66", "−0.60", "0.00"], ["G3V7U2", "Microtubule-associated protein 1 A, isoform CRA_c (Microtubule-associated protein 1A)", "0.65", "−0.62", "0.00"], ["P63041", "Complexin-1 (Complexin I) (CPX I) (Synaphin-2)", "0.62", "−0.69", "0.00"], ["F1LRZ7", "Neurofilament heavy polypeptide", "0.61", "−0.71", "0.01"], ["P01830", "Thy-1 membrane glycoprotein (Thy-1 antigen) (CD antigen CD90)", "0.57", "−0.81", "0.00"], ["G3V774", "F-box only protein 2 (F-box protein 2)", "0.56", "−0.83", "0.02"], ["P19527", "Neurofilament light polypeptide (NF-L) (68 kDa neurofilament protein) (Neurofilament triplet L protein)", "0.54", "−0.88", "0.01"], ["A0A0G2JZL9", "Endothelial cell-selective adhesion molecule", "0.53", "−0.90", "0.03"], ["A0A0G2K0T6", "Gamma-synuclein", "0.38", "−1.41", "0.00"], ["F1LUD3", "AHNAK nucleoprotein 2", "0.30", "−1.74", "0.01"]], "row_count": 65, "column_count": 5, "numeric_cells": 265, "header_rows": 1, "locator": "section[2]/table-wrap[2]#ijms-22-08592-t002"}}, {"id": "asset:table:pmid:34445296:3", "role": "table", "format": "jats-table", "media_type": "application/vnd.jats.table+json", "uri": null, "sha256": "sha256:8ba94c5336e9be6e94719b78561982871b500b9558d6c9ffd50bb2996d1b11a3", "bytes": 2879, "access": "open", "state": "verified", "reader": "read_table", "reader_status": "available", "identifier": "section[2]/table-wrap[3]#ijms-22-08592-t003", "version": null, "note": "Protein regulation in the nasal quadrant 2 weeks after pONT compared to contralateral control (p < 0.05 and FC log2 > 0.43 (upregulation) or ≤ −0.43 (downregulation)). R = right; L = left; FC = fold change.", "structure": {"pmcid": "PMC8395271", "section_index": 2, "section_title": "2. Results", "row_count": 28, "column_count": 5}, "table": {"label": "Table 3", "caption": "Protein regulation in the nasal quadrant 2 weeks after pONT compared to contralateral control (p < 0.05 and FC log2 > 0.43 (upregulation) or ≤ −0.43 (downregulation)). R = right; L = left; FC = fold change.", "rows": [["Uniprot", "Protein Name", "FC L/R", "LogFC", "p-Value"], ["Upregulation"], ["Q5U2P2", "Immunoglobulin superfamily member 11 (IgSF11)", "3.17", "1.67", "0.02"], ["D3ZZU8", "Fructosamine 3 kinase (Similar to fructosamine-3-kinase (Predicted), isoform CRA_a)", "2.47", "1.31", "0.02"], ["Q6MGB4", "H2-K region expressed gene 4, rat orthologue (RCG60794, isoform CRA_a) (Solute carrier family 39 (Zinc transporter), member 7) (Solute carrier family 39 member 7)", "2.07", "1.05", "0.00"], ["P47819", "Glial fibrillary acidic protein (GFAP)", "1.96", "0.97", "0.02"], ["Q5BJQ2", "Ubiquitin carboxyl-terminal hydrolase MINDY-1 (EC 3.4.19.12) (Deubiquitinating enzyme MINDY-1) (Protein FAM63A)", "1.86", "0.90", "0.01"], ["F1M118", "“Obsolete”", "1.85", "0.89", "0.02"], ["D3ZKK3", "Consortin, connexin sorting protein", "1.70", "0.77", "0.04"], ["D3ZPC4", "Neural cell adhesion molecule L1", "1.68", "0.75", "0.00"], ["Q6QI79", "Cap-specific mRNA (nucleoside-2′-O-)-methyltransferase 1 (LRRG00129)", "1.60", "0.68", "0.03"], ["F2Z3T8", "Guanine nucleotide-binding protein subunit gamma", "1.60", "0.68", "0.00"], ["F1M9D6", "Signal transducer and activator of transcription", "1.58", "0.66", "0.05"], ["P14056", "Serine/threonine-protein kinase A-Raf (EC 2.7.11.1) (Proto-oncogene A-Raf) (Proto-oncogene A-Raf-1)", "1.55", "0.63", "0.04"], ["O88775", "Embigin", "1.54", "0.62", "0.02"], ["Q6AYE2", "Endophilin-B1 (SH3 domain-containing GRB2-like protein B1)", "1.52", "0.61", "0.03"], ["G3V836", "Clusterin", "1.52", "0.60", "0.03"], ["P14480", "Fibrinogen beta chain (Liver regeneration-related protein LRRG036/LRRG043/LRRG189) (cleaved into: Fibrinopeptide B; Fibrinogen beta chain)", "1.51", "0.60", "0.01"], ["Q9EPF2", "Cell surface glycoprotein MUC18 (Gicerin) (Melanoma cell adhesion molecule) (Melanoma-associated antigen MUC18) (CD antigen CD146)", "1.47", "0.56", "0.02"], ["A0A0G2JUB0", "Protein FAM3C", "1.40", "0.49", "0.04"], ["P62193", "26S proteasome regulatory subunit 4 (P26s4) (26S proteasome AAA-ATPase subunit RPT2) (Proteasome 26S subunit ATPase 1)", "1.39", "0.48", "0.01"], ["M0R567", "Interferon regulatory factor 2-binding protein-like", "1.38", "0.46", "0.00"], ["Downregulation"], ["P63041", "Complexin-1 (Complexin I) (CPX I) (Synaphin-2)", "0.66", "−0.59", "0.02"], ["G3V7W1", "Programmed cell death protein 6 (Apoptosis-linked gene 2 protein homolog) (ALG-2)", "0.60", "−0.73", "0.01"], ["Q62813", "Limbic system-associated membrane protein", "0.56", "−0.82", "0.00"], ["D3ZDX5", "Plexin B1", "0.55", "−0.87", "0.04"], ["D4ABV0", "RGD1566320 (RGD1566320 (predicted))", "0.43", "−1.20", "0.01"]], "row_count": 28, "column_count": 5, "numeric_cells": 99, "header_rows": 1, "locator": "section[2]/table-wrap[3]#ijms-22-08592-t003"}}, {"id": "asset:table:pmid:34445296:4", "role": "table", "format": "jats-table", "media_type": "application/vnd.jats.table+json", "uri": null, "sha256": "sha256:5018a6d18643148d1f7bfdde1a43481c6a1999a6953e302d014584aae871e030", "bytes": 667, "access": "open", "state": "verified", "reader": "read_table", "reader_status": "available", "identifier": "section[2]/table-wrap[4]#ijms-22-08592-t004", "version": null, "note": "Bioinformatic analysis using SWATH-based proteomic data obtained from temporal (T) and nasal (N) retinal quadrants at 2 weeks after pONT compared to the corresponding contralateral control (T/N). R = right eye; L = left eye.", "structure": {"pmcid": "PMC8395271", "section_index": 2, "section_title": "2. Results", "row_count": 3, "column_count": 8}, "table": {"label": "Table 4", "caption": "Bioinformatic analysis using SWATH-based proteomic data obtained from temporal (T) and nasal (N) retinal quadrants at 2 weeks after pONT compared to the corresponding contralateral control (T/N). R = right eye; L = left eye.", "rows": [["", "Differentially Expressed Genes", "Genes with Measured Expression", "Gene Ontology TERMS", "miRNAs", "Upstream Regulators", "Chemical Upstream Regulators", "Associated Diseases"], ["LT vs. RT", "59", "2910", "842", "6", "72", "103", "27"], ["LN vs. RN", "24", "2901", "339", "0", "30", "72", "12"]], "row_count": 3, "column_count": 8, "numeric_cells": 14, "header_rows": 1, "locator": "section[2]/table-wrap[4]#ijms-22-08592-t004"}}, {"id": "asset:table:pmid:34445296:5", "role": "table", "format": "jats-table", "media_type": "application/vnd.jats.table+json", "uri": null, "sha256": "sha256:e2a819f34570f59811a74518de36a16a283d86cd1a3ec8531359da548647601a", "bytes": 846, "access": "open", "state": "verified", "reader": "read_table", "reader_status": "available", "identifier": "section[2]/table-wrap[5]#ijms-22-08592-t005", "version": null, "note": "Pathway analysis of regulated proteins in temporal and nasal quadrants 2 weeks after pONT.", "structure": {"pmcid": "PMC8395271", "section_index": 2, "section_title": "2. Results", "row_count": 13, "column_count": 4}, "table": {"label": "Table 5", "caption": "Pathway analysis of regulated proteins in temporal and nasal quadrants 2 weeks after pONT.", "rows": [["Pathway Name", "Pathway ID", "p-Value", "p-Value (FDR)"], ["LT vs. RT"], ["Ferroptosis", "04216", "5.332 × 10−4", "0.063"], ["HIF-1 signaling pathway", "04066", "0.002", "0.133"], ["Adipocytokine signaling pathway", "04920", "0.013", "0.527"], ["Necroptosis", "04217", "0.022", "0.594"], ["Cell adhesion molecules", "04514", "0.026", "0.584"], ["LN vs. RN"], ["Complement and coagulation cascades", "04610", "0.005", "0.401"], ["Hepatitis B", "05161", "0.018", "0.408"], ["Jak-STAT signaling pathway", "04630", "0.019", "0.408"], ["Pancreatic cancer", "05212", "0.024", "0.408"], ["Serotonergic synapse", "04726", "0.030", "0.408"]], "row_count": 13, "column_count": 4, "numeric_cells": 31, "header_rows": 1, "locator": "section[2]/table-wrap[5]#ijms-22-08592-t005"}}, {"id": "asset:table:pmid:34445296:6", "role": "table", "format": "jats-table", "media_type": "application/vnd.jats.table+json", "uri": null, "sha256": "sha256:01b33ac486da39fe72b337d676ba0be5f771f8b1da88136a14d508150266a6a6", "bytes": 2194, "access": "open", "state": "verified", "reader": "read_table", "reader_status": "available", "identifier": "section[2]/table-wrap[6]#ijms-22-08592-t006", "version": null, "note": "Gene ontology analysis of temporal and nasal quadrants 2 weeks after pONT.", "structure": {"pmcid": "PMC8395271", "section_index": 2, "section_title": "2. Results", "row_count": 17, "column_count": 7}, "table": {"label": "Table 6", "caption": "Gene ontology analysis of temporal and nasal quadrants 2 weeks after pONT.", "rows": [["Results", "LT vs. RT", "LN vs. RN"], ["GO Term", "p-Value", "p-Value (FDR)", "GO Term", "p-Value", "p-Value (FDR)"], ["Molecular functions", "Signaling receptor binding", "2.600 × 10−4", "0.054", "mRNA (nucleoside-2′-O-)-methyltransferase activity", "0.008", "0.222"], ["Protein-containing complex binding", "2.800 × 10−4", "0.054", "Fructosamine-3-kinase activity", "0.008", "0.222"], ["Thyroid hormone binding", "0.001", "0.130", "Cell adhesion molecule binding", "0.012", "0.222"], ["Structural constituent of postsynaptic intermediate filament cytoskeleton", "0.001", "0.130", "Signaling receptor binding", "0.013", "0.222"], ["Tau protein binding", "0.002", "0.130", "PDZ domain binding", "0.014", "0.222"], ["Biological processes", "Intermediate filament organization", "2.000 × 10−7", "5.018 × 10−4", "Homophilic cell adhesion via plasma membrane adhesion molecules", "2.700 × 10−4", "0.207"], ["Supramolecular fiber organization", "6.300 × 10−7", "5.854 × 10−4", "Positive regulation of multicellular organismal process", "6.900 × 10−4", "0.207"], ["Neuron projection regeneration", "7.000 × 10−7", "5.854 × 10−4", "Cell adhesion", "0.001", "0.207"], ["Intermediate filament-based process", "6.600 × 10−6", "0.002", "Biological adhesion", "0.001", "0.207"], ["Intermediate filament cytoskeleton organization", "6.600 × 10−6", "0.002", "Cell-cell adhesion via plasma-membrane adhesion molecules", "0.001", "0.207"], ["Cellular components", "Postsynaptic intermediate filament cytoskeleton", "3.400 × 10−5", "0.013", "Side of Membrane", "0.002", "0.310"], ["Neurofibrillary tangle", "8.400 × 10−5", "0.015", "External side of plasma membrane", "0.007", "0.310"], ["Axon", "1.700 × 10−4", "0.018", "Spherical high-density lipoprotein particle", "0.008", "0.310"], ["Schaffer collateral—CA1 synapse", "3.900 × 10−4", "0.018", "Cytoplasmic side of lysosomal membrane", "0.008", "0.310"], ["Neuronal cell body", "4.100 × 10−4", "0.018", "Perinuclear endoplasmic reticulum lumen", "0.008", "0.310"]], "row_count": 17, "column_count": 7, "numeric_cells": 62, "header_rows": 2, "locator": "section[2]/table-wrap[6]#ijms-22-08592-t006"}}], "gaps": ["asset:data:geo:PXD026783 not downloaded", "asset:data:geo:PXD026783 licence not read"]}, "analysis": {"state": "partial", "methods": [{"id": "method-1", "family": "regression_modelling", "specific": "ols", "label": "Regression modelling", "dsl_operations": ["ols_fit"], "missing_operations": [], "expressible": true, "parameters": {"r_squared": "S and MRM-MS (temporal, R2 = 0"}, "data_shapes": ["predictors_and_outcome"], "evidence": {"score": 0.7, "band": "medium", "quote": "Linear regression"}}, {"id": "method-2", "family": null, "specific": null, "label": "paired t-test", "dsl_operations": [], "missing_operations": [], "expressible": null, "parameters": {}, "data_shapes": [], "evidence": {"score": 1.0, "band": "verbatim", "quote": "Samples were normalized by most likely ratio (MLR) normalization with a paired t-test used to detect significantly differentiated proteins in biological samples among experimental groups."}}], "targets": [], "gaps": ["method.reader_named_unmapped"]}, "capability": {"state": "complete", "requirements": [{"capability_id": "capability:computational_analysis", "label": "Computational analysis platform", "assay_families": ["meta_analysis", "machine_learning", "survival_analysis", "statistical_regression", "pca", "molecular_docking", "network_analysis", "differential_expression", "gwas"], "availability_state": "owned", "tier_unlocked": "R1", "candidate": "Automated Assays Laravel application and simulated adapters"}], "gaps": []}, "readiness": {"state": "complete", "tier": "R1", "blocking_axis": "none", "blocked_on": "nothing:replicable_now", "axes": {"method": {"tier": "R1", "expressible": true, "required_operations": ["ols_fit"], "missing_operations": [], "basis": "The paper names the method it used and the corpus's method extractor maps it onto the DSL catalogue: ols."}, "capability": {"tier": "R1", "capability_id": "capability:computational_analysis", "availability_state": "owned", "basis": "The computational analysis is owned. 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