Sensitive and Deep Coverage Phosphoproteome Detection Method Reveals Spleen as a More Sensitive Organ than Liver in Early Detection of Liver Fibrosis-Related Signaling Protein Dysregulation

Although cathepsin S is transported from the spleen to the liver, where it cleaves collagen XVIII to produce endostatin and plays a critical role in the onset of early liver fibrosis, the relationship between liver fibrosis and spleen function remains underexplored. Given the roles of phosphorylation in disease, understanding its regulatory mechanism in early liver fibrosis is crucial. Despite advances in mass spectrometry enhancing phosphoproteomics, its application is limited by small clinical samples and subtle protein changes. We optimized a phosphoproteomic workflow, adjusting the protein amounts and using different enrichment beads and varied mass spectrometers, achieving deep phosphoproteomic coverage from minimal samples. We identified over 46,000 phosphosites in HepG2 cells and over 29,000 phosphosites in mouse liver samples using just 500 μg of proteins. Even with as little as 50 μg of 293T proteins, we detected over 11,000 phosphosites, 1.2 times more than the recently reported RUPE-phospho. Using the Sensitive and Deep Coverage Phosphoproteome Detection method, abbreviated as SDC-PhosDet, we demonstrated that in early liver fibrosis, the spleen exhibits more rapid and sensitive phosphorylation changes than the liver, affecting proteins closely linked to signaling and metabolism such as STAT1, JUN, CBL, ATP7B, and PTPN2. These findings highlight the spleen's role and offer new avenues for investigating the molecular mechanisms of early liver fibrosis, diagnosis, and intervention beyond the liver itself. Moreover, this method holds promise for applying phosphoproteomics to early-stage liver fibrosis using clinical microsamples.