Proteomic Analysis of Protein Ubiquitination Events in Dairy Goats with Fatty Liver
Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Sample Collection
2.2. Protein Extraction
2.3. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
2.4. Reductive Alkylation and Enzymatic Hydrolysis
2.5. Peptide Desalination and Quantification
2.6. Enrichment of Ubiquitinated Peptides
2.7. LC-MS/MS Data Acquisition
2.8. Database Search
2.9. Bioinformatics and Statistical Analysis
2.10. Histopathological Examination
2.11. Determination of TG Levels in Liver Tissue
2.12. Determination of Non-Esterified Fatty Acids (NEFA) and β-Hydroxybutyric Acid (BHBA) Levels in the Blood
2.13. Quantitative Real-Time PCR Analysis
3. Results
3.1. Histopathological Testing and TG Content Determination
3.2. Identification of Ubiquitin-Modified Proteins
3.3. Functional Enrichment of Ubiquitinated Proteins and Protein–Interaction Networks
3.4. Motif Analysis of the Identified and Quantifiable Proteins
3.5. Further Analysis of Ubiquitination in Hepatic Proteins
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Time (min) | B (%) |
---|---|
0 | 5 |
80 | 23 |
99 | 29 |
108 | 35 |
111 | 48 |
112 | 100 |
120 | Stop |
Item | Value |
---|---|
ProteomeDiscoverer version | 2.4 |
Protein Database | uniport-taxonomy-9925.unique.fasta |
Cys alkylation | Iodoacetamide |
Dynamic Midification | Oxidation (M), Acetyl (Protein N-Terminus), Acetyl(K), GG(K, S, T) |
Static Modification | Carbamidomethyl (C) |
Enzyme Name | Trypsin (Full) |
Max. Missed Cleavage Sites | 2 |
Precursor Mass Tolorance | 10 ppm |
Gene | Primer (5′–3′) | |
---|---|---|
Capra hircus ACSL5 | F | CGAAAATGGACTCTTGAC |
R | ACTCCTGGGTGTTCTCAT | |
Capra hircus ACSL1 | F | GGTTGACTTCCGGCAGTACG |
R | CCAGGTCGCATGGAGGCT | |
Capra hircus EHHADH | F | AGCGTGTCTTTGCTGAAC |
R | AGAATCGGCTGGGAATAA | |
Capra hircus ACAA1 | F | TGACGACAAGGGCACAGA |
R | CTTAAAGGCGGGCTTCAG | |
Capra hircus Actin | F | CTCAGAGCAAGAGAGGCAT |
R | CTCGTTGTAGAAGGTGTGGT |
Motif | Motif Core | Foreground | Background | Fold Increase | ||
---|---|---|---|---|---|---|
Matches | Size | Matches | Size | |||
xGxxxx_K_Gxxxxx | 10.55 | 20 | 1624 | 96 | 22,977 | 2.9 |
xxxxxx_K_Gxxxxx | 6.07 | 135 | 1604 | 1253 | 22,881 | 1.5 |
xxxGxG_K_xxxxxx | 6.89 | 22 | 1469 | 126 | 21,628 | 2.6 |
xxxxxx_K_xxxxxR | 3.64 | 106 | 1447 | 1106 | 21,502 | 1.4 |
xxxxxx_K_xxxRxx | 3.13 | 91 | 1341 | 91 | 20,396 | 1.4 |
xxxxxx_K_xxGxxx | 2.72 | 108 | 1250 | 108 | 19,418 | 1.3 |
xxxxIx_K_xxxxxx | 2.58 | 85 | 1142 | 85 | 18,156 | 1.4 |
xxxxxL_K_xxxxxx | 3.13 | 147 | 1057 | 147 | 17,168 | 1.3 |
xxGxxx_K_xxxxxx | 3.22 | 75 | 910 | 75 | 15,326 | 1.5 |
xxIxxx_K_xxxxxx | 2.38 | 60 | 835 | 60 | 14,471 | 1.4 |
xQxxxx_K_xxxxxx | 2.21 | 49 | 775 | 49 | 13,743 | 1.5 |
Protein | PepNumber | Delta p-Value (FL/Con) | Ubiquitination Sites |
---|---|---|---|
ACSL1 | 8 | −34.62495976 | K448; K284; K416; K572; K407; K377; K237; K532; K243 |
EHHADH | 15 | −22.19208675 | K330; K250; K222; K38; K591; K577; K584; K362; K171; K165; K535; K532; K700; K280; K318 |
ACAA1 | 3 | −16.61 | K395; K292; K198 |
ACSL5 | 3 | −16.61 | K412; K536; K616 |
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Zhu, Y.; Liu, Z.; Zhang, Y.; Meng, Y.; Song, X.; Li, J.; Zhang, Y.; Zhao, J.; Du, L.; Deng, Q. Proteomic Analysis of Protein Ubiquitination Events in Dairy Goats with Fatty Liver. Animals 2025, 15, 2010. https://doi.org/10.3390/ani15142010
Zhu Y, Liu Z, Zhang Y, Meng Y, Song X, Li J, Zhang Y, Zhao J, Du L, Deng Q. Proteomic Analysis of Protein Ubiquitination Events in Dairy Goats with Fatty Liver. Animals. 2025; 15(14):2010. https://doi.org/10.3390/ani15142010
Chicago/Turabian StyleZhu, Yuli, Zhenhua Liu, Yuming Zhang, Yao Meng, Xunuo Song, Jinyu Li, Yue Zhang, Junkang Zhao, Liyin Du, and Qinghua Deng. 2025. "Proteomic Analysis of Protein Ubiquitination Events in Dairy Goats with Fatty Liver" Animals 15, no. 14: 2010. https://doi.org/10.3390/ani15142010
APA StyleZhu, Y., Liu, Z., Zhang, Y., Meng, Y., Song, X., Li, J., Zhang, Y., Zhao, J., Du, L., & Deng, Q. (2025). Proteomic Analysis of Protein Ubiquitination Events in Dairy Goats with Fatty Liver. Animals, 15(14), 2010. https://doi.org/10.3390/ani15142010