Analysis of gangliosides directly from thin-layer chromatography plates by infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry with a glycerol matrix.
A novel method is presented for direct coupling of high-performance thin-layer chromatography (HPTLC) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the analysis of biomolecules. A first key feature is the use of a liquid matrix (glycerol), which provides a homogeneous wetting of the silica gel and a simple and fast MALDI preparation protocol. A second is the use of an Er:YAG infrared laser, which ablates layers of approximately 10-microm thickness of analyte-loaded silica gel and provides a soft desorption/ionization of even very labile analyte molecules. The orthogonal time-of-flight mass spectrometer employed in this study, finally provides a high accuracy of the mass determination, which is independent of any irregularity of the silica gel surface. The analytical potential of the method is demonstrated by the compositional mapping of a native GM3 (II(3)-alpha-Neu5Ac-LacCer) ganglioside mixture from cultured Chinese hamster ovary cells. The analysis is characterized by a high relative sensitivity, allowing the simultaneous detection of various major and minor GM3 species directly from individual HPTLC analyte bands. The lateral resolution of the direct HPTLC-MALDI-MS analysis is defined by the laser focus diameter of currently approximately 200 microm. This allows one to determine mobility profiles of individual species with a higher resolution than by reading off the chromatogram by optical absorption. The fluorescent dye primuline was, furthermore, successfully tested as a nondestructive, MALDI-compatible staining agent.
Institute of Medical Physics and Biophysics, Westfälische Wilhelms-Universität Münster, 48149 Münster, Germany. Klaus.Dreisewerd@uni-muenster.de
Klaus
K
Dreisewerd
Johannes
J
Müthing
Andreas
A
Rohlfing
Iris
I
Meisen
Zeljka
Z
Vukelić
Jasna
J
Peter-Katalinić
Franz
F
Hillenkamp
Stefan
S
Berkenkamp
D000818:Animals; D016466:CHO Cells; D002855:Chromatography, Thin Layer / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D005679:G(M3) Ganglioside / Q000737:chemistry* / Q000378:metabolism; D005990:Glycerol / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D005679:G(M3) Ganglioside; D005990:Glycerol