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Isobaric Labeling of Intact Gangliosides toward Multiplexed LC-MS/MS-Based Quantitative Analysis.


ABSTRACT: Gangliosides are sialic acid-containing glycosphingolipids recognized to play essential role in biological processes. Both the glycan and lipid structures influence their biological function and thus necessitate their determination as intact molecular species. To our knowledge, no multiplexed method for intact gangliosides currently exists. In this paper, we aimed to demonstrate an approach for isobaric labeling of intact gangliosides. Specifically, we carried out the rapid, chemoselective oxidation of sialic acid side chain in common ganglioside core structures using NaIO4 followed by ligation with a carbonyl-reactive isobaric tandem mass tag (TMT) reagent and subsequent RPLC-MS/MS analysis. Attachment of the isobaric label was observed to improve the ionization efficiency of complex gangliosides using electrospray ionization. Fragmentation of the resulting [M + 2H]2+ ions of TMT-labeled gangliosides provided information-rich spectra containing fragments from the glycan, lipid, and TMT reporter ions. This facile approach enabled simultaneous quantification of up to six samples as well as identification of glycan and lipid compositions in a single injection. As a proof-of-concept, using porcine brain total ganglioside extracts pooled at known ratios, we obtained overall sample-to-sample precision of <12% RSD and mean error of <10%. This showcased the great promise and feasibility of this strategy for high-throughput analysis of intact gangliosides in biological extracts.

PROVIDER: S-EPMC6069520 | BioStudies |

REPOSITORIES: biostudies

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