Fig. 7: Proteomics and N-terminomics/TAILS analyses of synovial fluids treated with buffer or tryptase β.

a Workflow of proteomics experimental design. Human synovial fluids from non-OA patients (n = 7) were incubated for 1 h with buffer or tryptase β at 1:100. Created using BioRender. b The numbers of unique and shared peptides between TAILS and preTAILS analysis. For a complete list, see Supplementary Tables 2–6. c The numbers of statistically changing peptides using an interquartile boxplot analysis between in the preTAILS samples. For a complete list, see Supplementary Table 2. d Left, Distribution of N-terminal peptides in the TAILS enrichment. Middle, statistically changing peptides using an interquartile boxplot analysis. Right, Distribution of post-translational peptide modifications as analyzed using TopFINDER⁹³. For a complete list, see Supplementary Tables 5, 6. e Left, peptide sequence profiles of significantly elevated neo-N-terminal peptides in tryptase β-treated synovial fluids identified in the TAILS analysis using IceLogo⁵⁸. f Left, peptide sequence profiles of significantly elevated neo-N-terminal peptides in buffer-treated synovial fluids identified in the TAILS analysis using IceLogo⁵⁸. Significantly (p < 0.05) overrepresented amino acids are shown above, and underrepresented residues are shown below the x-axis. Right, Cleavage sites identified tryptase β-treated synovial fluids are depicted as heat maps from P6 to P6′ residues. Green: Upregulated. Red: Downregulated. Statistical analysis was determined by a two-tailed unpaired Student’s t test and was adjusted for multiple comparisons. g) All identified PRG4 peptides. The ¹³³⁰K↓G¹³³¹ N-termini was significantly elevated in the Tryptase-treated samples. h STRING-db⁵⁹ analysis of N-termini elevated in the Tryptase-treated samples from Supplementary Table 6. An enrichment was detected for Toll-like receptor (red), Extracellular matrix organization (green), and Immune system (blue). Metascape⁴⁸ i pathway enrichment and j TRRUST analysis of the TAILS data and k) Metascape⁴⁸ analysis of the preTAILS data of different pathways between buffer- and tryptase β-treated synovial fluids. Accumulative hypergeometric p-values and enrichment factors were calculated and used for filtering as performed as a two-sided analysis (for i, j and k). Remaining significant terms were hierarchically clustered into a tree based on Kappa-statistical similarities among their gene’s memberships. Then, 0.3 kappa score was applied as the threshold to cast the tree into term clusters.