[[The]] characterization of bone extracellular matrix quality by proteome profiling, focusing on osteocalcin and glycation

Abstract

This study shows the development and execution of protocols that allow for a comparison of the proteome of healthy bone to fragile bone, and identifies specific post-translational modifications and/or the nature of such modifications in both collagenous (Type I collagen) and non-collagenous (osteocalcin) proteins associated with aging, altered microbiome and increased bone fragility.

Taken together, this study shows the development of a method to assess protein content of bone extracts, optimizing it for detection and qualification bone proteins proteome. Targets can then be assayed for a range of donors to establish a relationship between the protein concentration and bone health for aging and diseased bone.

Carboxymethyl-lysine (CML), an advanced glycation end-product that was irregularly detected in the analysis of whole bone proteome, was quantified by targeting specific parts of the proteome during protein extraction. CML content in bone showed correlation with increasing age and bone fragility.

Furthermore, the extraction technique was used to obtain high yields and quantify osteocalcin content of bone in response to varying microbiome status. The microbiome is a main source of vitamin K, which has a major role in the carboxylation of osteocalcin. Using mass spectroscopy, this study also shows that both glucose and ribose can post-translationally modify osteocalcin and such modifications, common in diabetes, localize on the n-terminal tyrosine of osteocalcin.

Osteoporosis is the most common human bone disease, associated with the increased susceptibility of bone to fracture. Osteoporosis is currently assessed using bone mineral density (BMD); however, recent studies have found normal or high BMD in bones with increased risk of fracture. This may be explained by the bone matrix quality, including the concentrations of collagenous and non-collagenous bone proteins and the post translational modifications of these proteins.

A protocol was developed and optimized to extract and identify collagenous and non-collagenous extracellular matrix, plasma and host of other proteins from bone through mass spectroscopy techniques (MALDI-TOF/TOF and LCMS). Efficacy of select proteins within bone proteome was shown to identify changes associated with aging and post-translationally modified bone and twenty-three different proteins with bone proteome were identified.