Quantitative phosphoproteomic analysis of mice with liver fibrosis by DIA mass spectrometry analysis with PRM verification.

Liver fibrosis (LF), commonly associated with chronic liver diseases, is a major public health problem worldwide. Protein phosphorylation is not only an important form of protein modification in organisms but also the most important mechanism to regulate and control the activity and function of proteins, affecting the occurrence and development of many diseases. However, comprehensive phosphoproteomic profiling in LF has not been fully elucidated. In this study, data-independent acquisition (DIA) was used to analyse the phosphoproteomics of mice with LF. A total of 553 phosphopeptides (representing 440 phosphoproteins) had significant phosphorylation levels. Among these phosphoproteins, 49 were upregulated and 401 were downregulated, and 5 phosphoserine (P-Ser) motifs and 2 phosphothreonine (P-Thr) motifs were conserved in LF. GO and KEGG pathway enrichment analyses identified 769 significant GO terms and 49 significant KEGG pathways. Four phosphorylated proteins were selected for parallel reaction monitoring (PRM) verification, and the results were consistent with DIA data. Together, there were significantly different phosphoproteomic profiles in LF, suggesting that protein phosphorylation was related to the occurrence and progression of LF, which could pave the way for further investigation into the related regulatory mechanisms. SIGNIFICANCE: LF is a necessary stage in the development of chronic liver disease to liver cirrhosis and has attracted wide attention. To the best of our knowledge, there are few reports on the phosphorylated proteomics of LF. In this study, DIA and PRM techniques were used to study the liver tissue of mice induced by CCl. The results showed that phosphorylation had a significant effect on the activity and function of proteins, and the PRM results were consistent with the trend observed in DIA analysis. This study will help to better reveal the relationship of phosphorylated proteins in LF and lay a foundation for further study of related regulatory mechanisms.