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Collagens form a common family of triple-helical proteins classified in 21 types. This unique structure is further stabilized by specific hydroxylation of distinct lysyl and prolyl residues forming 5-hydroxylysine and hydroxyproline (Hyp) isomers, mostly 4-trans and 3-trans-Hyp. The molecular distribution of the Hyp-isomers among the different collagen types is still not well investigated, even though disturbances in the hydroxylation of collagens are likely to be involved in several diseases such as osteoporosis and autoimmune diseases. Here, a new approach to analyze underivatized amino acids by hydrophilic interaction chromatography (HILIC) coupled on-line to electrospray ionization mass spectrometry (ESI-MS) is reported. This method can separate all three studied Hyp-isomers, Ile, and Leu, which are all isobaric, allowing a direct qualitative and quantitative analysis of collagen hydrolysates. The sensitivity and specificity was increased by a neutral loss scan based on the loss of formic acid (46 u).

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Control over crystal growth by acidic matrix macromolecules is an important process in the formation of many mineralized tissues. Highly acidic macromolecules are postulated intermediates in tissue mineralization, because they sequester many calcium ions and occur in high concentrations at mineralizing foci in distantly related organisms. A prerequisite for biomineralization is the ability of cations like calcium to bind to proteins and to result in concert with appropriate anions like phosphates or carbonates in composite materials with bone-like properties. For this mineralization process the proteins have to be modified with respect to acidification. In this study we modified the protein collagen by carboxymethylation using glucuronic acid. Our experiments showed unambigously, that N(epsilon)-carboxymethyllysine is the major product of the in vitro nonenzymatic glycation reaction between glucuronic acid and collagen. We hypothesized that the function of biomimetically carboxymethylated collagen is to increase the local concentration of corresponding ions so that a critical nucleus of ions can be formed, leading to the formation of the mineral. Thus, the self-organization of HAP nanocrystals on and within collagen fibrils was intensified by carboxymethylation.