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Posttranslational modifications of collagen studied by off‐line coupling of HPLC and CE
Author(s) -
Mikulíková Katerina,
Eckhardt Adam,
Mikšík Ivan
Publication year - 2006
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.200500398
Subject(s) - chemistry , chromatography , high performance liquid chromatography , glycation , maillard reaction , phosphoric acid , collagenase , lysine , biochemistry , peptide , enzyme , amino acid , organic chemistry , receptor
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 ε‐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×75 mm id) using 100 mM sodium 1‐heptanesulfonate in 100 mM phosphate buffer, pH 2.5 (NaH 2 PO 4 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.