Proteomic signature of human annulus fibrosus and cartilage endplate: divergent matrisomal architectures reveal complementary roles in intervertebral disc homeostasis.

PURPOSE: The baseline proteomic architecture of healthy human annulus fibrosus (AF) and cartilage endplate (CEP) is poorly defined. A rigorous healthy-tissue reference is essential for identifying the early molecular deviations that drive degenerative disc disease (DDD).

METHODS

AF (n = 20) and CEP (n = 21) tissues were harvested from healthy brain-dead organ donors (Pfirrmann Grade I). After 8 M urea/TEAB extraction, proteins were reduced, alkylated, and digested with sequencing-grade trypsin. Tryptic peptides were analysed in triplicate by nano-LC-MS/MS (Q-Exactive Plus Orbitrap) and processed with Proteome Discoverer 2.5 against UniProt Homo sapiens (FDR < 1%). Matrisome annotation used Human MatrisomeDB. GO and KEGG enrichment were used with DAVID and ShinyGO v0.82. Proteome overlap was quantified by Jaccard similarity; intra-tissue variability by Kruskal-Wallis analysis of log₂-normalised NSAF values.

RESULTS

470 proteins were identified in AF and 1,899 in CEP. The AF proteome was enriched in ECM glycoproteins (57% of matrisome), ECM regulators-notably serine protease inhibitors and matrix metalloproteinases (48%)-and glycolytic enzymes reflecting adaptation to hypoxia and tensile load. The CEP proteome featured higher collagen density (30%), ECM-affiliated proteins (48%), and extensive mitochondrial pathway enrichment (TCA cycle, oxidative phosphorylation), establishing it as a metabolically active interface for nutrient transport and proteostasis. AF-CEP proteome overlap was the lowest pairwise compartment comparison (≈ 20%), and CEP exhibited significantly greater intra-tissue variability than AF or NP (p = 2 × 10⁻³²).

CONCLUSION

This study delivers the first comprehensive paired proteomic atlas of healthy human AF and CEP. The AF emerges as a mechanically adaptive, ECM-remodelling tissue; the CEP as a metabolically specialised cartilage-bone interface. Integrated with the published healthy NP proteome, these data constitute a three-compartment human IVD molecular reference baseline for degeneration research and therapeutic target discovery.