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. 2011 Aug 15;305(2-3):138-150.
doi: 10.1016/j.ijms.2010.10.020.

Rapid Profiling of Bovine and Human Milk Gangliosides by Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

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Rapid Profiling of Bovine and Human Milk Gangliosides by Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Hyeyoung Lee et al. Int J Mass Spectrom. .
Free PMC article

Abstract

Gangliosides are anionic glycosphingolipids widely distributed in vertebrate tissues and fluids. Their structural and quantitative expression patterns depend on phylogeny and are distinct down to the species level. In milk, gangliosides are exclusively associated with the milk fat globule membrane. They may participate in diverse biological processes but more specifically to host-pathogen interactions. However, due to the molecular complexities, the analysis needs extensive sample preparation, chromatographic separation, and even chemical reaction, which makes the process very complex and time-consuming. Here, we describe a rapid profiling method for bovine and human milk gangliosides employing matrix-assisted desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS). Prior to the analyses of biological samples, milk ganglioside standards GM3 and GD3 fractions were first analyzed in order to validate this method. High mass accuracy and high resolution obtained from MALDI FTICR MS allow for the confident assignment of chain length and degree of unsaturation of the ceramide. For the structural elucidation, tandem mass spectrometry (MS/MS), specifically as collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) were employed. Complex ganglioside mixtures from bovine and human milk were further analyzed with this method. The samples were prepared by two consecutive chloroform/methanol extraction and solid phase extraction. We observed a number of differences between bovine milk and human milk. The common gangliosides in bovine and human milk are NeuAc-NeuAc-Hex-Hex-Cer (GD3) and NeuAc-Hex-Hex-Cer (GM3); whereas, the ion intensities of ganglioside species are different between two milk samples. Kendrick mass defect plot yields grouping of ganglioside peaks according to their structural similarities. Gangliosides were further probed by tandem MS to confirm the compositional and structural assignments. We found that only in human milk gangliosides was the ceramide carbon always even numbered, which is consistent with the notion that differences in the oligosaccharide and the ceramide moieties confer to their physiological distinctions.

Figures

Figure 1
Figure 1
MALDI-MS spectra of ganglioside GM3 from bovine buttermilk in (A) positive- and (B) negative ion detection mode. Zoom-in spectrum is shown in the inset. Symbols for the types of quasimolecular ions: ●, [M-H+2Na]+; ■, [M+Na]+; ▴, [M-CO2+Na]+. * Matrix-ganglioside adducts are detected with low abundance.
Figure 1
Figure 1
MALDI-MS spectra of ganglioside GM3 from bovine buttermilk in (A) positive- and (B) negative ion detection mode. Zoom-in spectrum is shown in the inset. Symbols for the types of quasimolecular ions: ●, [M-H+2Na]+; ■, [M+Na]+; ▴, [M-CO2+Na]+. * Matrix-ganglioside adducts are detected with low abundance.
Figure 2
Figure 2
MALDI-MS spectra of ganglioside GD3 from bovine buttermilk in (A) positive- and (B) negative ion detection mode. Symbols for the types of quasimolecular ions: ●, [M-2H+3Na]+; ■, [M-H+2Na]+; ▲, [M-H-H2O+2Na]+; ○, [M-H] ; □, [M+Na-2H]; ▵, [M-H2O-H].
Figure 2
Figure 2
MALDI-MS spectra of ganglioside GD3 from bovine buttermilk in (A) positive- and (B) negative ion detection mode. Symbols for the types of quasimolecular ions: ●, [M-2H+3Na]+; ■, [M-H+2Na]+; ▲, [M-H-H2O+2Na]+; ○, [M-H] ; □, [M+Na-2H]; ▵, [M-H2O-H].
Figure 3
Figure 3
Tandem MS spectra of ganglioside GM3 from bovine buttermilk in the positive mode using (A) CID and (B) IRMPD with the precursor ion at m/z 1295.794. Insets are the isolation of the peak of interest. (C) Fragmentation by tandem MS of the GM3 species. The structure depicted refers to GM3, the dominant form in bovine milk. The assignment of fragment ions is according to the nomenclature of Domon and Costello [32].
Figure 3
Figure 3
Tandem MS spectra of ganglioside GM3 from bovine buttermilk in the positive mode using (A) CID and (B) IRMPD with the precursor ion at m/z 1295.794. Insets are the isolation of the peak of interest. (C) Fragmentation by tandem MS of the GM3 species. The structure depicted refers to GM3, the dominant form in bovine milk. The assignment of fragment ions is according to the nomenclature of Domon and Costello [32].
Figure 4
Figure 4
Tandem MS spectra of ganglioside GD3 from bovine buttermilk in the positive mode using (A) CID and (B) IRMPD with the precursor ion at m/z 1608.872. (C) Fragmentation of the GD3 species detected at m/z 1608.872. The structure depicted refers to GD3 known as a dominant form in bovine milk.
Figure 4
Figure 4
Tandem MS spectra of ganglioside GD3 from bovine buttermilk in the positive mode using (A) CID and (B) IRMPD with the precursor ion at m/z 1608.872. (C) Fragmentation of the GD3 species detected at m/z 1608.872. The structure depicted refers to GD3 known as a dominant form in bovine milk.
Figure 5
Figure 5
Representative MALDI-MS spectra of gangliosides from bovine and human milk. Negative mode MALDI MS spectrum of (A) bovine milk gangliosides and (B) human milk gangliosides. Enlarged spectrum shows the ceramide heterogeneities of ganglioside GM3 species.
Figure 5
Figure 5
Representative MALDI-MS spectra of gangliosides from bovine and human milk. Negative mode MALDI MS spectrum of (A) bovine milk gangliosides and (B) human milk gangliosides. Enlarged spectrum shows the ceramide heterogeneities of ganglioside GM3 species.
Figure 6
Figure 6
Kendrick mass defect analysis of milk gangliosides. ◇, bovine milk ganglioside; ●, human milk ganglioside. (A) Kendrick mass plot, (B) GM3 cluster from human milk ganglioside, and (C) GD3 cluster from human milk ganglioside. Grouping of ions into distinct regions is shown in the Kendrick mass plot, and each cluster has several different degrees of unsaturation.
Figure 6
Figure 6
Kendrick mass defect analysis of milk gangliosides. ◇, bovine milk ganglioside; ●, human milk ganglioside. (A) Kendrick mass plot, (B) GM3 cluster from human milk ganglioside, and (C) GD3 cluster from human milk ganglioside. Grouping of ions into distinct regions is shown in the Kendrick mass plot, and each cluster has several different degrees of unsaturation.

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