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.
Joshi et al. (Mon,) studied this question.