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Separation and identification of GM1b pathway Neu5Ac- and Neu5Gc gangliosides by on-line nanoHPLC-QToF MS and tandem MS: toward glycolipidomics screening of animal cell lines.


Cell Line, Cell Separation, Chromatography, High Pressure Liquid, G(M1) Ganglioside, analogs & derivatives, chemistry, G(M3) Ganglioside, Glycolipids, Hybridomas, metabolism, Lipids, methods, Mice, N-Acetylneuraminic Acid, Nanotechnology, Neuraminic Acids, Spectrometry, Mass, Electrospray Ionization, Tandem Mass Spectrometry, Animals

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      Monosialoganglioside fraction of YAC-1 lymphoma cells was comprehensively analyzed and structurally defined by nano-high-performance liquid chromatography (nanoHPLC) in on-line conjunction with electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF MS). An efficient separation and sensitive detection of Neu5Gc-containing gangliosides from Neu5Ac-containing analogues was for the first time accomplished in a single nanoHPLC/ESI-QTOF MS run, as demonstrated for mouse hybridoma cell GM3 fraction containing GM3(Neu5Ac) and GM3(Neu5Gc) species and further applied for the analysis of YAC-1 lymphoma cell monosialoganglioside fraction. New insights into YAC-1 monosialoganglioside mixture heterogeneity were obtained: 31 distinct species, comprising 18 Neu5Gc-containing gangliosides and 13 Neu5Ac-containing species of GM1b and GalNAc-GM1b type were found to be expressed by YAC-1 cell line. On-line structural elucidation of individually separated Neu5Ac- and Neu5Gc-containing gangliosides provided strong evidence on the "GM1b-pathway" sourcing for monosialoganglioside synthesis. Such an analytical method is documented as superior to the classical approaches by increased speed of analysis, sensitivity and level of information, being thus a viable glycolipidomic tool.

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