Naslov
Analysis of Oligosaccharides and Gangliosides Directly from HPTLC Plates by IR-MALDI-o-TOF Mass Spectrometry with a Glycerol Matrix

Autori
Dreisewerd, Klaus ; Muething, Johannes ; Pohlentz, Gottfried ; Rohlfing, Andreas ; Meisen, Iris ; Vukelić, Željka ; Kölbl, Stefanie ; Peter-Katalinić, Jasna ; Hillenkamp, Franz ; Berkenkamp, Stefan

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
53rd ASMS Conference on Mass Spectrometry Abstracts / American Society for Mass Spectrometry - San Antonio (TX) : American Society for Mass Spectrometry, 2005

Skup
53rd ASMS Conference on Mass Spectrometry

Mjesto i datum
San Antonio (TX), Sjedinjene Američke Države, 05.06.2005. - 09.06.2005

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
Oligosaccharides; Gangliosides; HPTLC; IR-MALDI-o-TOF; Glycerol Matrix

Sažetak
We present a novel method for the direct coupling of high-performance thin-layer chromatography (HPTLC) with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) for the analysis of glycoconjugates. One key feature of the method is the use of glycerol as a liquid matrix, providing an efficient wetting of the silica gel. A second is the use of an Er:YAG IR-laser, which ablates small volumes of analyte-loaded silica gel and provides a soft desorption/ionization of analyte molecules. The employed orthogonal time-of-flight mass spectrometer (o-TOF-MS), finally, provides a high mass accuracy, independent of any irregularity of the silica gel surface. The potential of the method is demonstrated by the compositional mapping of a native, singly sialylated ganglioside mixture (GM3) from CHO cells and by a mixture of native oligosaccharides from human milk. HPTLC plates (200 μ m-silica gel film ; glass backing) were from Merck and used as delivered. Gangliosides (containing exclusively Neu5Ac) were purified in-house and separated by HPTLC using chloroform/methanol/ water (120/85/20, v/v/v), supplemented with 2 mM CaCl2 as the developing system. Oligosaccharides were separated by developing the HPTLC plate twice in n-butanol/acetic acid/water (110:45:45, v/v/v) in a saturated chamber with intermediate drying. After development, HPTLC plates were cut into stripes and fixed on a milled-out MALDI-target. Analyte bands were identified by a parallel orcinol-stained run. Glycerol was applied onto the unstained run by either spotting volumes of 0.2 - 0.5 μ l onto identified bands or by spraying a glycerol-methanol mixture over the entire chromatogram. The employed mass spectrometer was a modified Sciex o-TOF prototype, equipped with an additional IR-laser. The focal laser spot size was on the order of 5 x 10-2 mm2. 100-200 laser shots were applied for acquisition of the mass spectra displayed in Figures 1-3. The orcinol-stained reference chromatogram of the ganglioside mixture is shown in the inset of Figure 1. The GM3 mixture separates into two distinct bands. The lower band contains GM3 species with a C16:0 fatty acid residue as the major compound. The upper band contains GM3 species with longer fatty acids, mainly C22:0, C24:1, and C24:0, as indicated in the figure. The arrow indicates the direction of the chromatographic mobility. The sphingoid base residue is d18:1 for all GM3 species. Two direct HPTLC-IR-MALDI mass spectra, acquired from the upper analyte band in positive and negative ion mode are displayed in Figure 1. In positive ion mode, gangliosides are essentially recorded as doubly sodiated ions as the base peaks. For the major species, a low abundance of asialo-fragments was also detected (a few % of the intensity of the intact ion ; data not shown). In negative ion mode, gangliosides are detected as [M-H]- ions as the base peaks, and a fragmentation of the molecular ions was not notable (data not shown). However, adduct formation with glycerol and NaCl complicates the mass spectrum. In both ion modes, a series of GM3 species with minor abundance is found next to the major GM3 components. Experimental and calculated m/z-values agree within 50 ppm for all species. The limit of detection was determined by a dilution series to about 50 ng of total GM3 sample applied for HPTLC. Additional experiments showed that the notable occurrence of the C16:0 species in the upper HPTLC band (next to its presence as the major component in the lower one) is not due to diffusion in the glycerol matrix. The orcinol-stained reference chromatograms of a human milk oligosaccharide sample (HM-10), prefractionated by size exclusion chromatography, is displayed in Figure 2. The expected identities of oligosaccharide components are indicated next to the three analyte bands. The third band (Fuc2-LNT) produced only a faint optical contrast. Three mass spectra recorded in positive ion mode from an unstained chromatogram at the indicated positions are displayed in Figure 2. Only 0.6 μ g of total oligosaccharide sample were applied in this case for HPTLC, which is a factor of ten less than the 6 μ g used for the reference chromatogram. As expected, lacto-N-tetraose (LNT), singly fucosylated LNT, and doubly fucosylated LNT are recovered by HPTLC-MALDI-MS in the three bands. The oligosaccharides are detected as singly sodiated species as the base peaks, accompanied by a few glycerol or sodiumhydrogenphosphate adduct ions of lower intensity. The approximate limit of detection was determined in a dilution series to about 1 pmol of an individual oligosaccharide species applied for HPTLC. Figure 3 finally shows a „ 2-D“ HPTLC-MS plot, acquired by scanning the laser across the analyte bands. The center-to-center distance of the irradiated spots was ~ 300 μ m. The width of the milk oligosaccharide bands as determined by MS corresponds well to one determined from the optically stained reference lane. Financial Support by the Deutsche Forschungsgemeinsschaft under grant no. DR416/5-1 is gratefully acknowledged.

Izvorni jezik
Engleski

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