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.

No abstract

The application of a recently developed direct coupling of high-performance thin-layer chromatography (HPTLC) and infrared matrix-assisted laser desorption/ionization orthogonal extracting time-of-flight mass spectrometry (Dreisewerd, K.; Müthing, J.; Rohlfing, A.; Meisen, I.; Vukelic, Z.; Peter-Katalinic, J.; Hillenkamp, F.; Berkenkamp, S. Anal. Chem. 2005, 77, 4098-4107) to the analysis of phospholipid mixtures is demonstrated. Mixtures of six phospholipid types were exemplarily analyzed. The sensitivity was found to be in the range between about 10 and 150 pmol of material spotted for HPTLC, depending on phospholipid acidity, Rf value, and ion polarity. The lateral resolution of the analysis is on the order of the laser focus diameter of about 220 x 300 microm2, allowing differentiation between phospholipid species of different acyl chain composition within one single HPTLC band, which were undistiguishable by a mere visual assessment. Analyte diffusion due to the addition of glycerol to the HPTLC plate was found to be-if at all notable-of only minor importance.

A fast piezoelectric detection system was utilized to record time-resolved photoacoustic signals reflecting the thermal expansion and the rate of material ablation in infrared matrix-assisted laser desorption/ionization (IR-MALDI). Glycerol was employed as a liquid and 2,5-dihydroxybenzoic acid (2,5-DHB) as a crystalline matrix. An Er:YAG laser (λ ) 2.94 µm; τ L ∼ 100 ns) and a wavelength-tunable optical parametric oscillator (OPO) laser system (λ ) 1.4-4.0 µm; τ L ) 6 ns) were used for desorption/ionization. Material ejection and MALDI ion signals were recorded as a function of laser fluence, pulse duration, and wavelength. For glycerol as matrix, material ejection was found to be temporally confined to about the laser pulse duration when the OPO was employed. For excitation with the Er:YAG laser, a prolonged material ejection with an approximately exponential decay was observed with a characteristic time constant of ∼1 µs. For both lasers, material ejection was observed already at fluences substantially below the MALDI ion threshold. For excitation with the OPO, a correlation between a change in the desorption/ablation mechanism and the onset of ion generation was found. Crystalline 2,5-DHB preparations were investigated with the OPO only. Material ejection from this matrix was found to exhibit a significantly different dependence of the overall ejected material on laser fluence. The article also provides an introduction to the underlying photoacoustic theory adapted to the MALDI process.