Biochemical Characterization of Major Bone-Matrix Proteins Using Nanoscale-Size Bone Samples and Proteomics Methodology

Abstract

There is growing evidence supporting the need for a broad scale investigation of the proteins and protein modifications in the organic matrix of bone and the use of these measures to predict fragility fractures. However, limitations in sample availability and high heterogeneity of bone tissue cause unique experimental and/or diagnostic problems. We addressed these by an innovative combination of laser capture microscopy with our newly developed liquid chromatography separation methods, followed by gel electrophoresis and mass spectrometry analysis. Our strategy allows indepth analysis of very limited amounts of bone material, and thus, can be important to medical sciences, biology, forensic, anthropology, and archaeology. The developed strategy permitted unprecedented biochemical analyses of bone-matrix proteins, including collagen modifications, using nearly nanoscale amounts of exceptionally homogenous bone tissue. Dissection of fully mineralized bone-tissue at such degree of homogeneity has not been achieved before. Application of our strategy established that: (1) collagen in older interstitial bone contains higher levels of an advanced glycation end product pentosidine then younger osteonal tissue, an observation contrary to the published data; (2) the levels of two enzymatic crosslinks (pyridinoline and deoxypiridinoline) were higher in osteonal than interstitial tissue and agreed with data reported by others; (3) younger osteonal bone has higher amount of osteopontin and osteocalcin then older interstitial bone and this has not been shown before. Taken together, these data show that the level of fluorescent crosslinks in collagen and the amount of two major noncollagenous bone matrix proteins differ at the level of osteonal and interstitial tissue. We propose that this may have important implications for bone remodeling processes and bone microdamage formation.

Age-related changes in the plasticity and toughness of human cortical bone at multiple length scales

“…Whereas we believe that increased nonenzymatic cross-linking plays a prime role in suppressing deformation at the fibrillar level (22,33,39,40), at this size scale, additional age-related changes to the organic matrix (3,(43)(44)(45)(46), the mineral size (47), and posttranslational modifications to the collagen (23) may influence the material properties. Additionally, variability in both mineralization (48)(49)(50) and the local mineral content (51-53) due to disease or osteoporosis treatments are known to affect the mechanical properties and may play a role in aging.…”

Assessment of collagen quality associated with non-enzymatic cross-links in human bone using Fourier-transform infrared imaging

“…The osteons are surrounded by interstitial, lamellar bone, which essentially reflect parts of former osteons and therefore predate the current osteons. Thus, some areas of the bone microstructure have an older tissue age ( e.g., interstitial tissue) and a younger tissue age ( e.g., newly formed osteons) [45]. Similar remodeling processes occur in trabecular bone.…”

Bone protein extraction without demineralization using principles from hydroxyapatite chromatography

“…Protein concentration was determined using Coomassie (Bradford) Assay Kit (Thermo-Scientific) with BSA as a protein standard, and all samples were desalted using micro dialysis (3500 MWCO regenerated cellulose; Fisher Scientific) against nanopure water [32] for 4 days.…”