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Introduction A Disintegrin-like And Metalloprotease with Thrombospondin type 1 Motifs 8 (ADAMTS8) is a secreted multidomain enzyme active against extracellular matrix (ECM) proteins. ADAMTS8 is primarily expressed in the lung and its overexpression has been recently implicated in pulmonary arterial hypertension (PAH). In a mouse model of hypoxia-induced PAH, mice with a targeted deletion of Adamts8 in pulmonary arterial smooth muscle cells (Adamts8ΔSM22α ) showed lower right ventricular (RV) systolic pressure and less RV hypertrophy compared to wild-type (WT) mice. However, ADAMTS8’s precise role in PAH, including its substrate repertoire, remains unclear. To date, osteopontin, a multifunctional and highly phosphorylated ECM protein, is the only identified substrate. Here, a degradomics approach was applied to identify ADAMTS8 substrates with potential relevance to PAH.Methods Candidate substrates were identified using N-Terminal Amine Isotopic Labelling of Substrates (N-TAILS) proteomics. Human full length ADAMTS8 WT and its catalytically inactive mutant E365→Q (EQ) variant, both with a C-terminal FLAG tag, were expressed in HEK293T cells, followed by anti-FLAG affinity purification. Expression levels were validated using Coomassie Brilliant Blue staining and anti-FLAG immunoblotting. Serum-free conditioned media from fibroblasts was collected, concentrated, and digested with ADAMTS8 WT, with ADAMTS8 EQ serving as control. The digested proteins were reduced, alkylated, and then labelled with heavy or light formaldehyde. The reaction was quenched with Tris and digested with trypsin. A small sample was reserved for pre-TAILS analysis. The remaining peptides were enriched using polyglycerol-aldehyde polymer, which binds to trypsin-generated (unlabelled) termini, thereby enriching for labelled N-termini. The non-bound peptides were filtered to remove the polymer and obtain the N-TAILS sample. TAILS and pre-TAILS fractions were analysed on a TIMS-TOF Bruker instrument and data were processed with FragPipe/MSFragger, followed by Perseus and Excel.Results ADAMTS8 WT and its catalytically inactive EQ variant were successfully expressed and purified. Coomassie Brilliant Blue staining confirmed the purity of the batches. N-TAILS proteomics identified several ADAMTS8 substrates potentially relevant to PAH and fibrosis including fibronectin, and collagen I and VI.Conclusion These results suggest that the ADAMTS8 degradome includes ECM proteins associated with PAH and fibrosis, offering further insights into its role in PAH and supporting its potential as a therapeutic target.