B subunit monomers of cholera toxin bind G1 ganglioside class as revealed by chip- nanoelectrospray multistage mass spectrometry

The noncovalent interaction between the monomers (Ctb) of the B subunit (Ctb5) of cholera toxin from Vibrio cholerae and a native complex mixture of gangliosides extracted and purified from adult human cerebellum was studied using an analytical platform encompassing fully automated chip- nanoelectrospray ionization (nanoESI) on a NanoMate robot coupled to a high-capacity ion trap (HCT) mass spectrometer (MS). The interaction assay involved the incubation of Ctb5 and gangliosides dissolved in 10mM ammonium acetate buffer at pH 5.8. This pH value enhanced the dissociation of the monomers constituting Ctb5 and allowed the direct interaction between the monomers and gangliosides. Aliquots of the reaction products were collected directly after 10, 30, and 60min of incubation in the 96-well plate of the NanoMate robot and immediately submitted to MS analysis. Except for the Ctb envelope, positive ion mode chip-nanoESI mass spectra presented 25 signals corresponding to multiply protonated ions of the Ctb complexes with different gangliosides, all belonging to the G1 class. Certain complexes revealed Ctb interaction with G1 species of high sialylation degree or modified by fucosylation, which were not discovered in human cerebellum before. Multistage fragmentation carried out by electron transfer dissociation (ETD) followed by collision-induced dissociation (CID) on the Ctb complex with Fuc-GT1 (d18:1/18:0) provided data supporting the binding of the c isomer of Fuc-GT1 (d18:1/18:0) to histidine 14 (H14), most probably via Neu5Ac.