High-sensitivity analysis of glycosphingolipids by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight imaging mass spectrometry on transfer membranes

Abstract

Glycosphingolipids are ubiquitous constituents of cells. Yet there is still room for improvement in the techniques for analyzing glycosphingolipids. Here we report our highly sensitive and convenient analytical technology with imaging mass spectrometry for detailed structural analysis of glycosphingolipids. We were able to determine detailed ceramide structures; i.e., both the sphingosine base and fatty acid, by MS/MS/MS analysis on a PVDF membrane with 10 pmol of GM1, with which only faint bands were visible by primuline staining. The limit of detection was approximately 1 pmol of GM1, which is lower than the value in the conventional reports (10 pmol).

Fig. 1. Molecular structures of GM1 used in this study

Abbreviations correspond to the nomenclature of Svennerholm et al.[59]. It has been shown that major molecular species of GM1 from the bovine brain were d18:1/18:0 shown in (A) and d20:1/18:0 shown in (B)[57,58].

Fig. 2. Imaging mass spectrometry of GM1 by TLC-Blot-MALDI-QIT-TOF MS

(A) The overall spectrum obtained from a GM1 band on the PVDF membrane. (B) An optical image of GM1 on thin-layer chromatogram stained with primuline. (C) and (D) Ion images obtained from GM1 of different ceramide moieties, m/z 1572 and 1544, respectively. (E) Ion images were merged with m/z 1572 shown in red and m/z 1544 in green. The arrow shows the direction of the raster scan.

Fig. 3. Distributional analysis of GM1 by TLC-Blot-MALDI-QIT-TOF MS

(A) The densitometric analysis of imaging data acquired from the raster scan of the GM1 band on the PVDF membrane (red, m/z 1572; green, m/z 1544). The X-axis indicates the distance from the top to the bottom pixel. The arrow shows the direction of the raster scan. (B) Semi-quantitative mass spectra acquired from the area with asterisks in (A). Each area of the rectangles with asterisks shows a parallel ratio between m/z 1572 and m/z 1544 in the mass spectrum with the same number of asterisks. (*; m/z 1572 was mainly observed, **; m/z 1572 and 1544 were equally detected, ***; m/z 1544 was mainly observed.)

Fig. 4. TLC-Blot-MALDI-QIT-TOF MS spectra of primuline-stained GM1 on the PVDF membrane (negative ion mode)

(A) Thin-layer chromatogram stained with primuline. Lanes 1–8 contain bovine brain GM1: (lane 1–2) 1 nmol, (lane 3–4) 100 pmol, (lane 5–6) 10 pmol, and (lane 7–8) 1 pmol. The plate was developed with chloroform/methanol/0.2% aqueous CaCl₂, (55/45/10, v/v/v), and GM1 spots were detected with primuline staining then transferred to a PVDF membrane. Each GM1 was directly analyzed by TLC-Blot-MALDI-QIT-TOF MS. (B), (C), (D), and (E) are spectra derived from 1 nmol (lane 1), 100 pmol (lane 3), 10 pmol (lane 5), and 1 pmol (lane 7) of GM1, respectively. The detection limit of GM1 was estimated at 1 pmol. The number of laser irradiations was 2 shots in arbitrarily 100 spots per band.

Fig. 5. Nomenclature for cleavages of precursor ions of ceramides

Annotations reported by Ann et al. are cited[60].

Fig. 6. TLC-Blot-MALDI-QIT-TOF MS, MS/MS, and MS/MS/MS spectra of GM1 to identify ceramide moieties

(A) MS spectrum; (B) MS/MS spectrum obtained with the precursor ion at m/z 1544; (C) MS/MS/MS spectrum obtained with the second precursor ion at m/z 564. (D) MS/MS spectrum obtained with the precursor ion at m/z 1572; (E) MS/MS/MS spectrum obtained with the second precursor ion at m/z 592.

Fig. 7. Sensitivity test of TLC-Blot-MALDI-QIT-TOF MS for the analysis of the ceramide structure at 10 pmol of GM1

(A)MS spectrum; (B) MS/MS spectrum obtained with the precursor ion at m/z 1544; (C) MS/MS/MS spectrum obtained with the second precursor ion at m/z 564. The detection limit of TLC-Blot-MALDI-QIT-TOF MS/MS/MS for structural identification of the ceramide moiety was found to be almost 10 pmol.

Fig. 8. TLC-Blot-MALDI-QIT-TOF MS analyses of GSLs extracted from a Tay-Sachs patient

(A) Thin-layer chromatogram stained with primuline: (lane 1) GSLs standards (GM1, GD1a, GD1b, and GT1b); (lane 2) GSLs derived from the brain of a patient with Tay-Sachs disease and pooled to produce fraction X. The plate was developed with chloroform/methanol/0.2% aqueous CaCl₂, (55/45/10, v/v/v), and spots were detected on the TLC plate by primuline staining and were then transferred onto a PVDF membrane. (a), (b), (c), and (d) represent the spectra derived from the band produced by fraction X, and (e) represents the spectrum corresponding to the GD1a standard. Each band was directly analyzed by TLC-Blot-MALDI-QIT-TOF MS and bands (a), (b), (c), and (d) were identified as sulfatide, GM2, GM1, and GalNAc-GD1a, respectively.