Deciphering the Intratissue-Specific Collagen PTM Site-Specific Heterogeneity in Human Adrenal Extracellular Matrix

Adrenal glands are one of the pivotal glands of the human endocrine system. Recently, the extracellular matrix (ECM) of the adrenal capsule and cortex was explored in two fractions: outer fraction (OF) and inner fraction (IF). A significant variation in the ECM proteins' levels, including collagens, was documented. Collagen undergoes a plethora of post-translational modifications (PTMs), exhibiting crucial roles such as cell-matrix interaction and cross-linking. However, the site-specific identification and characterization of collagen PTMs remain challenging and are unknown for the human adrenal gland. By applying our in-house proteomics pipeline, we identified 1044 4-hydroxyproline (4-HyP), 205 3-hydroxyproline (3-HyP), 106 hydroxylysine (HyK), 17 galactosyl-hydroxylysine (G-HyK), and 37 glucosyl galactosyl-hydroxylysine (GG-HyK) sites from abundant collagen chains of both fractions. Although the site-specificity of collagen PTMs (3-HyP, HyK, and G/GG-HyK) across fractions is conserved, the occupancies were different in a site-specific manner. The microheterogeneity observed in K⁸⁶² of COL1A1 demonstrated that the hydroxylation level was higher in OF, while glycosylation levels were higher in IF. This points toward a change in the cross-linking of collagen I across both fractions. Furthermore, our analysis revealed that site-specific O-glycosylation, particularly GG-HyK, in basement membrane collagen-IV is higher, which may contribute to the secretion of steroids from the adrenal gland. In this study, we have annotated collagen PTMs, developed a COL1A1 PTM map, and quantitated site-specific PTMs in the human adrenal gland. Taken together, this work reveals the intratissue-specific site-specific collagen PTM heterogeneity although the ECM is stable, which is noteworthy, and lays the foundation for understanding their role in region-specific functions.