Coupling porous sheathless interface MS with transient-ITP in neutral capillaries for improved sensitivity in...

IgG antibodies are modulated in their function by the specific structure of the N-glycans attached to their Fc (fragment crystallizable) portions. However, the glycosylation analysis of antigen-specific IgGs is a challenging task as antibody levels to a given antigen only represent a fraction of the total IgG levels. Here, we investigated the use of a transient-ITP (t-ITP)--MS method for highly sensitive IgG1 glycosylation profiling as a complementary method to a high-throughput nano-RPLC-MS method. It was found that t-ITP-CZE using neutrally coated separation capillaries with a large volume injection (37% of capillary volume) and interfaced to MS with a sheathless porous sprayer yielded a 40-fold increase in sensitivity for IgG1 Fc glycopeptide analysis when compared to the conventional strategy. Furthermore, the glycoform profiles found with the t-ITP-CZE strategy were comparable to those from nano-RPLC-MS. In conclusion, the use of the highly sensitive t-ITP-CZE-MS method will provide information on IgG Fc glycosylation for those samples with IgG1 concentrations below the LODs of the conventional method.