Fast characterization of industrial soy protein isolates by direct analysis with matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Industrial soy protein isolates (SPIs) due to differences in their processing conditions may differ both in composition and in degree of hydrolysis. As a result, they display different performance in food production and final food properties like consistency and taste. To address this issue, a fast, cheap, and simple method for screening and characterization is required. In this article, the successful analysis of soy protein isolates, a complex mixture of proteins with glycinin and beta-conglycinin as major components, by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is demonstrated. The preparation implements a fast extraction of the proteins from the raw SPI either under neutral or reducing conditions. The extracts are analyzed subsequently by MALDI-TOF-MS without further purification. Results of the two conditions are compared. Finally, different SPIs from different suppliers are analyzed and compared concerning their consistency. The method could be applied to other plant proteins and mixtures thereof. Since the composition and intactness of different subunits play important roles in functional properties of soy proteins, rapid methods for fingerprinting of different industrial soy protein sources will be valuable tools for successful product formulation.

Principles of laser-induced separation and transport of living cells.

Separation and transport of defined populations of living cells grown on a thin membrane can be achieved by laser microdissection (LMD) of the sample of interest, followed by a laser-induced forward transport process [laser pressure "catapulting" (LPC)] of the dissected cell cluster. We investigate the dynamics of LMD and LPC with focused and defocused UV-A laser pulses by means of time-resolved photography. Catapulting is driven by plasma formation when tightly focused pulses are used, and by confined thermal ablation at the bottom of the sample for defocused catapulting. With both modalities, the initial specimen velocity amounts to about 50 to 60 ms. Time-resolved photography of live cell catapulting reveals that in defocused catapulting, strong shear forces arise when the sample is accelerated out of the culture medium covering the cells. By contrast, pulses focused at the periphery of the specimen cause a fast rotational movement that minimizes the flow of culture medium parallel to the sample surface, and thus the resulting shear stresses. Therefore, the recultivation rate of catapulted cells is much higher when focused pulses are used. Compared to collateral damage by mechanical forces, side effects by heat and UV exposure of the cells play only a minor role.

Mechanisms of laser-induced dissection and transport of histologic specimens.

Rapid contact- and contamination-free procurement of histologic material for proteomic and genomic analysis can be achieved by laser microdissection of the sample of interest followed by laser-induced transport (laser pressure catapulting). The dynamics of laser microdissection and laser pressure catapulting of histologic samples of 80 mum diameter was investigated by means of time-resolved photography. The working mechanism of microdissection was found to be plasma-mediated ablation initiated by linear absorption. Catapulting was driven by plasma formation when tightly focused pulses were used, and by photothermal ablation at the bottom of the sample when defocused pulses producing laser spot diameters larger than 35 microm were used. With focused pulses, driving pressures of several hundred MPa accelerated the specimen to initial velocities of 100-300 m/s before they were rapidly slowed down by air friction. When the laser spot was increased to a size comparable to or larger than the sample diameter, both driving pressure and flight velocity decreased considerably. Based on a characterization of the thermal and optical properties of the histologic specimens and supporting materials used, we calculated the evolution of the heat distribution in the sample. Selected catapulted samples were examined by scanning electron microscopy or analyzed by real-time reverse-transcriptase polymerase chain reaction. We found that catapulting of dissected samples results in little collateral damage when the laser pulses are either tightly focused or when the laser spot size is comparable to the specimen size. By contrast, moderate defocusing with spot sizes up to one-third of the specimen diameter may involve significant heat and ultraviolet exposure. Potential side effects are maximal when samples are catapulted directly from a glass slide without a supporting polymer foil.

Principles of laser microdissection and catapulting of histologic specimens and live cells.

Rapid contact- and contamination-free procurement of specific samples of histologic material for proteomic and genomic analysis as well as separation and transport of living cells can be achieved by laser microdissection (LMD) of the sample of interest followed by a laser-induced forward transport process [laser pressure "catapulting," (LPC)] of the dissected material. We investigated the dynamics of LMD and LPC with focused and defocused laser pulses by means of time-resolved photography. The working mechanism of microdissection was found to be plasma-mediated ablation. Catapulting is driven by plasma formation, when tightly focused pulses are used, and by ablation at the bottom of the sample for moderate and strong defocusing. Driving pressures of several hundred megapascals accelerate the specimen to initial velocities of 100-300 m/s before it is rapidly slowed down by air friction. With strong defocusing, driving pressure and initial flight velocity decrease considerably. On the basis of a characterization of the thermal and optical properties of the histologic specimens and supporting materials used, we calculated the temporal evolution of the heat distribution in the sample. After laser microdissection and laser pressure catapulting (LMPC), the samples were inspected by scanning electron microscopy. Catapulting with tightly focused or strongly defocused pulses results in very little collateral damage, while slight defocusing involves significant heat and UV exposure of up to about 10% of the specimen volume, especially if samples are catapulted directly from a glass slide. Time-resolved photography of live-cell catapulting revealed that in defocused catapulting strong shear forces originate from the flow of the thin layer of culture medium covering the cells. By contrast, pulses focused at the periphery of the specimen cause a fast rotational movement that makes the specimen wind its way out of the culture medium, thereby undergoing much less shear stresses. Therefore, the recultivation rate of catapulted cells was much higher when focused pulses were used.

Localization of noncovalent complexes in MALDI-preparations by CLSM.

The unambiguous detection of noncovalent complexes (NCCs) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is still a far cry from being routine. For protein NCCs such as their quaternary structure it has been reported that signals of the intact complex are only obtained for the first or at most the first few laser exposures of a given sample area. This observation was called the first-shot phenomenon. In the present study, this first-shot phenomenon has been investigated for the hexameric protein complex allophycocyanine (APC) by two independent methods, MALDI-MS with a (nearly) pH-neutral matrix 6-aza-2-thiothymine (6-ATT) and by imaging the fluorescence of the complex in APC-6-ATT preparations by confocal laser scan microscopy (CLSM). The intact APC heterohexamer loses its visible fluorescence upon dissociation into its subunits. Both methods consistently show that intact APC complexes are precipitated at the matrix crystal surface, but dissociate upon incorporation into the matrix crystals.

MALDI-TOF-MS analysis of bacterial spores: wet heat-treatment as a new releasing technique for biomarkers and the influence of different experimental parameters and microbiological handling.

Short wet heat-treatment is presented as a new technique to release high-mass biomarkers to obtain strain-specific fingerprints of intact bacterial spores by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Wet heat-treatment was applied for several minutes (3-30) by two techniques using either a screw-cap tube submerged in a glycerol bath at 120 degrees C or an Eppendorff-tube submerged in a water bath at 100 degrees C. Both techniques turned out to be successful for releasing high-mass biomarkers. The influence of different experimental parameters and microbiological handling on the peak pattern of the released high-mass biomarkers was studied. While the sporulation medium, the applied washing procedure, and the choice of matrix crucially influenced the peak pattern, other parameters like storage conditions were found to be insignificant. A protocol of optimized experimental conditions for MALDI-MS of wet heat-treated spores is presented.