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 NaIO 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] 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.

Biological Functions and Analytical Strategies of Sialic Acids in Tumor

“…A rapid periodate oxidation of the sialic acid side chain in common core structures of gangliosides was carried out using NaIO 4 treatment followed by ligation with a carbonyl-reactive isobaric tandem mass tag (TMT) and subsequent LC-MS/MS analysis. The TMT tag improved the ionization efficiency of complex gangliosides and could simultaneously quantify up to six samples, as well as the identification of glycan and lipid compositions in a single injection [129]. For N-glycans, triplex mdSUGAR tags were developed for quantitative glycomics.…”

Gangliosides are essential endosomal receptors for quasi-enveloped and naked hepatitis A virus

“…Gangliosides equivalent to 100 μg of protein from Huh-7.5 cell lysates were oxidized using mild periodate oxidation and labeled with aminoxyTMT 6-plex reagent (Thermo Scientific Pierce) as previously described 15 . Briefly, gangliosides were reconstituted in 250 μL of oxidation buffer (0.1 M sodium acetate/acetic acid, pH ~5.5) and added to 12.5 μL of 1 mM sodium metaperiodate (NaIO 4 ) in oxidation buffer.…”

“…The mixture was incubated on ice for 1 hr in the dark before quenching with 2.5 μL of 100 mM aqueous glycerol and further incubation for 1 hr on ice. The mixture was then subjected to C18 solid-phase extraction and the eluates collected, dried under a stream of nitrogen gas, and labelled with aminoxyTMT following the manufacturer’s instructions with minor modifications 15 . Briefly, the labeling reagent was dissolved in 200 μL of 95% methanol with 0.1% acetic acid, vortexed, and mixed with oxidized gangliosides for 10 min at room temperature.…”

“…To understand which specific ganglioside(s) might function in HAV entry, we utilized a recently developed, multiplexed LC-MS/MS assay 15 to characterize gangliosides present in miglustat-treated and untreated Huh-7.5 cells. Fourteen specific species representing 5 classes of gangliosides were identified: GM3 (16% of the total), GM2 (72%), GM1 (4.8%), GD3 (5.7%) and GD1a (0.6%) ( Fig.…”

O-acetylated Gangliosides as Targets for Cancer Immunotherapy

“…Isobaric labeling of gangliosides by chemoselective oxidation of the sialic acid side chain, followed by oxydation and ligation with a carbonyl reactive tandem mass tag reagent on C9 generates fluorescent gangliosides, which can be identified and quantified using Reverse-Phase Liquid Chromatography Mass Spectrometry (RPLC-MS). RPLC-MS allows the detection and quantification of GM1a, GD1a and GT1b as major brain porcine gangliosides [62]. Rossdam and coworkers have set up a multiplex Capillary Gel Electrophoresis coupled to Laser Induced Fluorescence detection (xCGE-LIF) to analyze carbohydrate moieties of gangliosides after ceramidase digestion.…”