Identifying Endogenous Proteins of Perennial Ryegrass (Lolium perenne) with Ex Vivo Antioxidant Activity
Abstract
:1. Introduction
2. Materials and Methods
2.1. Grass Cultivation and Wet Fractionation
2.2. Crude Protein Estimation and Dry Matter Analysis
2.3. One-Dimensional SDS-PAGE
2.4. Protein Fractionation by Size Exclusion Chromatography
Size Estimation of Protein Fractions
2.5. DPPH Radical Scavenging Activity Screening and Fraction Selection
2.6. Quantification of Antioxidant Properties in Selected Protein Fractions
2.6.1. Desalting and Protein Concentration Determination
2.6.2. Protein Concentration
2.6.3. DPPH Radical Scavenging Activity
2.6.4. Iron Chelation Activity
2.6.5. EC50 Calculations
2.7. Bottom-Up Proteomics by LC-MS/MS
2.7.1. In-Solution Digest of Selected SEC Fractions
2.7.2. Protein Extraction and In-Solution Digest of Crude Fractions
2.7.3. LC-MS/MS Analysis
2.7.4. LC-MS/MS Data Processing
2.7.5. Downstream Data Analysis of MaxQuant Data from SEC Fractions
Isoform Combination
Gene Ontology Analysis and Data Filtering
2.7.6. Downstream Data Analysis of Crude Fractions
2.8. Statistical Analysis
3. Results and Discussion
3.1. Protein Characterization of Wet Fractionation
3.2. Protein Fractionation and Selection
3.3. Ex Vivo Antioxidant Activity
3.4. Overview of Protein Composition and Most Abundant Proteins
3.5. Prediction of Antioxidant Proteins Using GO-Term Analysis
3.6. Correlating Protein Abundance and GO-Term Analysis with In Vitro Antioxidant Activity
3.7. Abundance of Known Antioxidant Proteins in Crude Fractions
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
Appendix B
Appendix C
Appendix D
References
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Crude Fraction | Mass (g) | DM (% w/w) | DM Distribution | Crude Protein 1 (%, DM Basis) | Crude Protein Distribution |
---|---|---|---|---|---|
Grass | 75.2 | 19.2 ± 1.1 a | 100% | 11.2 ± 0.4 a | 100% |
Pulp | 32.9 | 27.7 ± 3.1 b | 63.0% | 10.4 ± 0.4 a | 58.7% |
Green Juice | 34.4 | 15.5 ± 2.1 a | 36.9% | 12.5 ± 0.5 b | 41.0% |
DPPH | Iron Chelation | ||||
---|---|---|---|---|---|
Protein Fraction | MWavg (kDa) | EC50 (μg/mL) | EC50 (μM) | EC50 (μg/mL) | EC50 (μM) |
3 | n.d. 1 | 130 | n.d. 1 | n.d. 2 | n.d. 1,2 |
5 | 407 | 120 | 0.29 | n.d. 2 | n.d. 2 |
7 | 278 | 150 | 0.53 | 6.5 | 0.023 |
16 | 50.0 | n.d. 2 | n.d. 2 | 9.0 | 0.18 |
19 | 28.2 | n.d. 2 | n.d. 2 | 7.4 | 0.26 |
22 | 15.9 | n.d. 2 | n.d. 2 | 5.9 | 0.37 |
25 | 9.00 | n.d. 2 | n.d. 2 | 7.0 | 0.78 |
28 | 5.08 | n.d. 2 | n.d. 2 | 7.2 | 1.4 |
34 | 1.62 | 191 | 120 | 4.8 | 3.0 |
37 | 0.914 | 63 | 69 | 0.57 | 0.62 |
39 | 0.624 | 38 | 61 | 0.44 | 0.71 |
43 | 0.291 | 51 | 170 | 2.1 | 7.1 |
48 | 0.112 | 28 | 250 | 1.6 | 14 |
51 | 0.0634 | 13 | 210 | 0.60 | 9.5 |
55 | 0.030 | 26 | 880 | 0.31 | 11 |
Trolox | 0.251 | 37 ± 18 | 150 ± 74 | ||
EDTA | 0.292 | 2.5 ± 1.5 | 8.5 ± 5.1 |
Protein | Lead AC#s | Cat. | Mechanism | Ref. |
---|---|---|---|---|
ATP synthase (α, β, and γ subunits) | A8Y9G7 A8Y9H7 I1GU08 | N | Formation of ATP | [54] |
ATPase (V-type α subunit) | I1GVU2 | N | Formation of ADP by ATP hydrolysis | [55] |
Thioredoxin-dependent peroxiredoxin (TPx) | I1IXL1 I1IAA5 | A | Reduction of hydrogen peroxide and hydroperoxides. | [56,57] |
Superoxide dismutase (SOD) | I1I9J4 | A | Oxygen radical scavenging | [58] |
Ferredoxin-NADP reductase (FNR) | I1H1Z5 I1HW30 | I | NADPH regeneration | [59] |
Lactoylglutathione lyase (Glyoxalase I, Glo1) | A0A0Q3HLX8 | I | Glutathione formation | [60] |
L-ascorbate peroxidase (APX) | H6BDN2 | A | Reduction of hydrogen peroxide | [61] |
Glutaredoxin-dependent peroxiredoxin (GPx) | I1HY81 | A | Reduction of hydrogen peroxide | [62] |
Glutathione S-transferase (GST) | A0A165FYU2 | I | Reduction of lipid hydroperoxides by facilitating GSH binding | [63] |
Peroxidase (Px) | I1HZ46 | A | Reduction of hydrogen peroxide | [64] |
Putative dehydroascorbate reductase (DHAR) | H6BDN5 | I | Regeneration of ascorbate | [65] |
Peroxiredoxin Q-like (ycf33) 1 | A0A0Q3H912 | A | Reduction of alkyl hydroperoxides | [66] |
Ribulose-phosphate 3-epimerase (RPE) | I1H9A1 | N | Calvin cycle enzyme | [67] |
Sedoheptulose-1,7-biphosphatase (SBP) | I1HTG2 | N | Calvin cycle enzyme | [68] |
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Pedersen, K.D.A.; Andersen, L.T.; Heiselberg, M.; Brigsted, C.A.; Støvring, F.L.; Mikkelsen, L.M.; Hansen, S.A.; Rusbjerg-Weberskov, C.E.; Lübeck, M.; Gregersen Echers, S. Identifying Endogenous Proteins of Perennial Ryegrass (Lolium perenne) with Ex Vivo Antioxidant Activity. Proteomes 2025, 13, 8. https://doi.org/10.3390/proteomes13010008
Pedersen KDA, Andersen LT, Heiselberg M, Brigsted CA, Støvring FL, Mikkelsen LM, Hansen SA, Rusbjerg-Weberskov CE, Lübeck M, Gregersen Echers S. Identifying Endogenous Proteins of Perennial Ryegrass (Lolium perenne) with Ex Vivo Antioxidant Activity. Proteomes. 2025; 13(1):8. https://doi.org/10.3390/proteomes13010008
Chicago/Turabian StylePedersen, Kathrine Danner Aakjær, Line Thopholm Andersen, Mads Heiselberg, Camilla Agerskov Brigsted, Freja Lyngs Støvring, Louise Mailund Mikkelsen, Sofie Albrekt Hansen, Christian Enrico Rusbjerg-Weberskov, Mette Lübeck, and Simon Gregersen Echers. 2025. "Identifying Endogenous Proteins of Perennial Ryegrass (Lolium perenne) with Ex Vivo Antioxidant Activity" Proteomes 13, no. 1: 8. https://doi.org/10.3390/proteomes13010008
APA StylePedersen, K. D. A., Andersen, L. T., Heiselberg, M., Brigsted, C. A., Støvring, F. L., Mikkelsen, L. M., Hansen, S. A., Rusbjerg-Weberskov, C. E., Lübeck, M., & Gregersen Echers, S. (2025). Identifying Endogenous Proteins of Perennial Ryegrass (Lolium perenne) with Ex Vivo Antioxidant Activity. Proteomes, 13(1), 8. https://doi.org/10.3390/proteomes13010008