The use of 2-dimensional CNBr peptide maps for the analysis of crosslinked peptides in bone collagen

CNBr peptides from insoluble bovine cortical bone collagen were analyzed using a 2-D mapping technique. The major type 1 collagen CNBr peptides were detected by fluorography after general-labelling with 3H-NaBH4 in dimethyl-formamide. These maps were similar to those visualized by coomassie blue staining and demonstrated a proportional decrease of alpha 1CB6. New groups of peptides, different from those normally present in soluble type I collagen were detected. Some of these peptides were slightly larger and more acidic than alpha 1CB6 and were highly labelled when the demineralized bone was specifically labelled for the presence of aldehydes and crosslinks with 3H-NaBH4 in a phosphate buffer, pH 7.4. Based on the size and charge characteristics of these specifically labelled peptides, they were tentatively identified as crosslinking peptides containing different combinations of alpha 1CB6, alpha 1CB0,1 and alpha 1CB5. The specificity of the labelling method using 3H-NaBH4 in phosphate buffer was demonstrated by the detection of other known crosslinked peptides and by the virtual absence of label in alpha 1CB7, CB8, and CB3. We feel that this simple methodological approach developed in these experiments will prove to be very useful in the analysis of collagen crosslinks present in insoluble collagens derived from normal tissues of various ages as well as from pathological states.