Isomeric separation of permethylated glycans by extra-long reversed-phase liquid chromatography (RPLC)-MS/MS
Junyao Wang, Xue Dong, Aiying Yu, Yifan Huang, Wenjing Peng, Yehia Mechref
Abstract
-glycan isomers derived from model glycoproteins, including bovine fetuin, ribonuclease B and κ-casein. Baseline separations were observed on multiple sialylated linkage isomers. However, the separation performance of high-mannose isomers needs further improvement. The reproducibility and stability of this long C18 column was also tested by doing run-to-run, day-to-day and month-to-month comparisons of retention times on multiple glycans and the %RSD was found less than 0.92%. Finally, we applied this approach to separate glycan isomers derived from complex biological samples, including blood serum and cell lines, where baseline separations were attained on several isomeric structures. Compared to the separation efficiency of PGC and MGC columns, the RPLC C18 column provides lower resolution but more robust reproducibility, which makes it a good complementary alternative for isomeric separations of glycans.