Proteomic Atlas of Healthy Human Annulus Fibrosus and Cartilage Endplate Reveals Divergent Matrisomal Architectures

The intervertebral disc (IVD), critical for spinal flexibility and load bearing, is frequently affected by degenerative disc disease (DDD), a major cause of back pain. A significant gap exists in understanding the baseline proteomic architecture of healthy human annulus fibrosus (AF) and cartilage endplate (CEP). This lack of a rigorous healthy-tissue reference hinders the identification of early molecular deviations that initiate and drive DDD pathogenesis, making it challenging to develop targeted preventative or therapeutic strategies.

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

The study identified 470 proteins in the AF and 1,899 proteins in the CEP, revealing vastly different molecular compositions. The AF proteome was significantly enriched in ECM glycoproteins (57% of matrisome) and ECM regulators (48%), notably serine protease inhibitors and matrix metalloproteinases, alongside glycolytic enzymes, reflecting its adaptation to hypoxia and tensile load. In contrast, the CEP proteome featured a higher collagen density (30%), ECM-affiliated proteins (48%), and extensive mitochondrial pathway enrichment, including the TCA cycle and oxidative phosphorylation, establishing it as a metabolically active interface crucial for nutrient transport and proteostasis.

The AF-CEP proteome overlap was the lowest pairwise compartment comparison, at approximately 20%, highlighting their distinct biological roles. Furthermore, CEP exhibited significantly greater intra-tissue variability than AF or NP (p = 2 × 10⁻³²).