Posttranslational modifications of collagen studied by off-line coupling of HPLC and CE.
A typical example of non-enzymatic change of collagen is glycation (the Maillard reaction, formation of advanced glycation end products) resulting from the reaction of sugars with the epsilon-amino group of lysine. Posttranslational non-enzymatic modifications of collagen by sugars were studied. Collagenous tissues were incubated as a test protein separately with both glucose and ribose. The collagen mixture was digested by bacterial collagenase and separated by reversed-phase HPLC (in a Jupiter Proteo 90 A column). The eluate from this HPLC separation was collected as seven fractions and consecutively analysed by CE in a bare fused silica capillary (57/50 cm x 75 mm id) using 100 mM sodium 1-heptanesulfonate in 100 mM phosphate buffer, pH 2.5 (NaH2PO4 adjusted to pH by phosphoric acid). The chromatographic and electromigration behaviour of individual peptides varied considerably. This off-line HPLC-CE coupling made it possible to discover minor changes in the structure of collagen caused by posttranslational modifications. A new HPLC-CE technique for peptide analysis was developed, and applied to the identification of posttranslational modifications in slowly metabolised test proteins.
Mikulíková
Katerina
K
Institute of Physiology, Czech Academy of Science, Prague, Czech Republic. katka.mikulikova@seznam.cz
Eckhardt
Adam
A
Miksík
Ivan
I
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D015416:Maillard Reaction; D008297:Male; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D051381:Rats; D017208:Rats, Wistar; D012266:Ribose / Q000737:chemistry
D012266:Ribose; D003094:Collagen