Specific detection of glycopeptides using glycan diagnostic ions: a key step for the characterization of the glideosome from T. gondii.

摘要

The protein extract enriched in glycoproteins was digested and analyzed by nanoLC-MS/MS on the ion trap using parameters optimized in the model study. A glycopeptide (136-143) from Myosin XIV (one of the subunit involved in the penetration mechanism of the parasite) was identified and carries on Asn136 an oligomannosidic chain. Heterogeneity was also characterized and MS/MS data confirmed the presence of the following two high mannose structures, Man_(7)GlcNAc_(2) or Man_(8)GlcNAc_(2) on the peptide NYTSEALR. We have showed that the best results for detection of glycopeptides have been obtained by generating glycan diagnostic ions using in source collisions rather than fragmentation in the collision cell. For the glycopeptides characterization in a complex glycoprotein digest we have used a two step method: 1) LC-MS with specific diagnostic ions detection to determine the elution of minor glycopeptides, 2) LC-MS/MS optimized for the transmission of glycopeptides molecular ions and the detection of large fragment ions.