Proteomics of Collagen Pep tides: A Method to Reveal Minor Changes in Post-Translationally Modified Collagen by HPLC and Capillary Electrophoresis

摘要

Depository effects in slowly metabolized proteins, typically glycation or the estimation of products arising from the reaction of unsaturated long chain fatty acid metabolites (possessing aldehydic groups) are very difficult to assess owing to their extremely low concentration in the protein matrix. Posttranslational nonenzymatic modifications of collagen with sugars and oxidation products of lipid metabolism in tail tendons of two groups of rats (controls and hypertriglyceridemics with a high fructose diet) were studied. Collagen (a mixture of type I and III) was digested by bacterial collagenase and separated by reversed-phase HPLC; the profile obtained was divided into five fractions, which were further characterizated by capillary electrophoresis (CE) in a bare fused silica capillary (37/30 cm x 75 |xm ID). As the chroma-tographic and electromigration behaviour of individual peptides was considerably different, the combination of HPLC and CE appeared as a suitable approach capable of acceptable separation of complex peptide mixtures (over 150 peptide peaks were obtained on the electropherograms). This two-stepped peptide mapping with subsequent statistical evaluation (linear regression analysis) was shown to represent a reliable approach for revealing posttranslational modifications in slowly metabolized model proteins in vivo.