Figure 5.

EIC of representative oxidized ganglioside GT1b d18:1/18:0 (A) before labeling (panel 1), residual oxidized species left after labeling (panel 2) and the resulting aminoxyTMT⁰ labeled product (panel 3) where notable enhancement in ionization is apparent. Representative MS/MS spectrum of the [M+2H]²⁺ of aminoxyTMT⁰-labeled GM1a d18:1/18:0 (B). The TMT reporter ion region of (B) is magnified to show the presence of interfering peak from GalNAc (m/z 126.0550) and its baseline resolution with the reporter ion m/z 126.1280; data were acquired at 15,000 resolution setting at MS₂ level using Q Exactive HF (C); Δ indicates absolute mass error. Also observed is the contribution of the isotopic [M+1] peak of the aminoxyTMT label at m/z 127.1315. Reporter ion region of the [M+H]⁺ of unlabeled GM1a d18:1/18:0 (D) (for reference, the full MS/MS fragmentation of [M+H]⁺ ion of the unlabeled ganglioside is shown in Supporting Information Fig. S6b). This indicates that the most significant interfering ion for the analysis of gangliosides using the aminoxyTMT labeling in positive ion mode is the GalNAc-derived oxonium ion as described in the present work.