Phosphoproteomic analysis reveals differential associations between liver-spleen disharmony and qi-blood deficiency syndromes in chronic fatigue syndrome.

backgroundChronic fatigue syndrome (CFS) is a debilitating disorder characterized by persistent fatigue that is not alleviated by rest and is often accompanied by multiple somatic symptoms. The etiology of CFS remains poorly understood, and conventional Western medicine offers limited effective targeted therapies. In contrast, Traditional Chinese Medicine (TCM), which utilizes pattern differentiation-particularly the Liver-Spleen Disharmony Pattern (LSDP) and the Qi-Blood Deficiency Pattern (QBDP)-has demonstrated clinical efficacy in managing CFS. However, the molecular mechanisms underpinning TCM pattern classification in CFS remain largely unexplored.

methodsA total of 30 participants were enrolled in this study, including 10 CFS patients with LSDP, 10 CFS patients with QBDP, and 10 age- and sex-matched healthy controls (HC). Serum phosphoproteomic profiling was conducted using liquid chromatography-tandem mass spectrometry (LC-MS/MS), which incorporated data-dependent acquisition (DDA) for spectral library construction and data-independent acquisition (DIA) for label-free quantification. Differentially phosphorylated sites (DPSs) and proteins (DPPs) were identified with thresholds of absolute fold change (|FC|) ≥ 1.2 and a Benjamini-Hochberg (BH)-corrected false discovery rate (FDR) < 0.05. Principal component analysis (PCA) was employed to assess global differences in phosphorylation profiles across groups, and functional enrichment analyses were performed to elucidate the biological functions of differential molecules.

resultsPCA revealed distinct clustering of phosphoproteomic profiles among the three groups, with high consistency across biological replicates (PC1 explained 19.3% of the total variance, and PC2 explained 15.9%). A total of 849 non-redundant DPSs and 586 non-redundant DPPs were identified across the three pairwise comparisons. The HC vs. LSDP comparison yielded the highest number of differential molecules (406 DPSs and 351 DPPs), with a balanced distribution of upregulated and downregulated events. In contrast, the HC vs. QBDP comparison was dominated by phosphorylation upregulation (61.2% of DPSs), while the QBDP vs. LSDP comparison showed a higher proportion of downregulated DPSs (56.7%). Functional enrichment analysis indicated that upregulated DPPs in the HC vs. LSDP comparison were primarily involved in MAPK signaling and cytoskeletal remodeling, while downregulated DPPs were enriched in pathways associated with neurodegenerative diseases and nucleocytoplasmic transport. Notably, we identified a candidate differential phosphorylation site, DENND3 S472 (S472@DENND3_HUMAN), with moderate discriminatory power (raw p = 0.042, BH-corrected FDR < 0.05, AUC = 0.72).

conclusionThis exploratory study identified significant differences in serum phosphoproteomic profiles between CFS patients with LSDP and QBDP. The distinct phosphoproteomic signatures observed in LSDP and QBDP provide preliminary molecular evidence supporting TCM pattern differentiation in CFS. These findings enhance the understanding of CFS pathogenesis and lay the groundwork for precision-based TCM diagnosis and individualized therapeutic strategies for CFS.