Combination of high-performance anion-exchange chromatography and electrospray mass spectrometry for analysis of the in vitro O-glycosylated mucin motif peptide.

Reversed-phase high-performance liquid chromatography (HPLC) and high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection were developed for the study of products obtained from the in vitro O-glycosylation of a mucin motif peptide, TTSAPTTS, the most representative sequence encoded by the human gene MUC5C. After incubation of the peptide, which is rich in clustered hydroxyamino acids, by both human colonic and gastric microsomal homogenates, the glycosylated products were separated by HPLC and HPAEC and analysed by electrospray mass spectrometry (ES-MS). The combination of HPAEC and ES-MS was the approach used for evaluating the differences between the polypeptide N-acetylgalactosaminyltransferase activity in different digestive tissues.