Quantitation of collagen types I, III and V in tissue slices by capillary electrophoresis after cyanogen bromide solubilization
Abstract
A method for the determination of the proportions of major fiber-forming collagens (types I, III and V) in soft connective tissue was elaborated. The method is based on the release of insoluble collagen by CNBr with subsequent separation of the arising peptides.
For routine application the peptides are separated by capillary electrophoresis (50 mM phosphate pH 2.5, 15 kV, 50 degrees C, 70/60 cmX70 mu m I.D. capillary with UV detection at 200 nm). Quantitation of collagen type I can be done either on the basis of spiking the sample with a peptide mixture obtained from a known amount of collagen type I, or by spiking the sample with an equimolar mixture of the two peptides [alpha(1)(I)CB2 and alpha(1)(I)CB4] (constituting a fused peak) along with alpha(1)(III)CB2 and alpha(1)(V)CB1.
Compared to the previously published methods the procedure is faster and does not require isolation of marker peptides by tedious chromatographic procedures in a preceding preparatory step. Good results are obtained within a wide range of run buffer concentrations and applied voltages; conversely, intensive cleaning of the capillary after every three runs is recommended with a new capillary after 20-30 runs.