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Klaus Dreisewerd

Publications and source records attributed to Klaus Dreisewerd.

9 recordsLinked to original sources
PubMed2006

New insights into the glycosylation of the surface layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a.

The surface of Geobacillus stearothermophilus NRS 2004/3a cells is covered by an oblique surface layer (S-layer) composed of glycoprotein subunits. To this S-layer glycoprotein, elongated glycan chains are attached that are composed of [-->2)-alpha-l-Rhap-(1-->3)-beta-l-Rhap-(1-->2)-alpha-L-Rhap-(1-->] repeating units, with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain and a core saccharide as linker to the S-layer protein. On sodium dodecyl sulfate-polyacrylamide gels, four bands appear, of which three represent glycosylated S-layer proteins. In the present study, nanoelectrospray ionization time-of-flight mass spectrometry (MS) and infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry were adapted for analysis of this high-molecular-mass and water-insoluble S-layer glycoprotein to refine insights into its glycosylation pattern. This is a prerequisite for artificial fine-tuning of S-layer glycans for nanobiotechnological applications. Optimized MS techniques allowed (i) determination of the average masses of three glycoprotein species to be 101.66 kDa, 108.68 kDa, and 115.73 kDa, (ii) assignment of nanoheterogeneity to the S-layer glycans, with the most prevalent variation between 12 and 18 trisaccharide repeating units, and the possibility of extension of the already-known -->3)-alpha-l-Rhap-(1-->3)-alpha-l-Rhap-(1--> core by one additional rhamnose residue, and (iii) identification of a third glycosylation site on the S-layer protein, at position threonine-590, in addition to the known sites threonine-620 and serine-794. The current interpretation of the S-layer glycoprotein banding pattern is that in the 101.66-kDa glycoprotein species only one glycosylation site is occupied, in the 108.68-kDa glycoprotein species two glycosylation sites are occupied, and in the 115.73-kDa glycoprotein species three glycosylation sites are occupied, while the 94.46-kDa band represents nonglycosylated S-layer protein.

Bacillaceae↗
PubMed2006

Analysis of native milk oligosaccharides directly from thin-layer chromatography plates by matrix-assisted laser desorption/ionization orthogonal-time-of-flight mass spectrometry with a glycerol matrix.

We have recently presented a new method for direct coupling of high-performance thin-layer chromatography (HPTLC) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), illustrated by the analysis of a complex ganglioside mixture. In the current communication, an adaptation of this procedure to mixtures of native oligosaccharides from human and from elephant milk is described. The key features in this method are (1) glycerol as a liquid matrix, to provide a homogeneous wetting of the silica gel and a simple and fast MALDI preparation protocol, (2) an infrared (IR) laser for volume material ablation and particular soft desorption/ionization conditions, and (3) an orthogonal time-of-flight mass spectrometer for a high mass accuracy, independent of any irregularity of the silica gel surface. Chromatographic "mobility profiles" were determined by scanning the laser beam across the analyte bands. The current limit of detection for the MS analysis was determined to approximately 10 pmol of individual oligosaccharides spotted for chromatography. A liquid composite matrix, containing glycerol and the ultraviolet (UV-)MALDI matrix alpha-cyano-4-hydroxycinnamic acid, allows a direct HPTLC-MALDI-MS analysis with a 337 nm-UV laser as well. Compared to the IR-MALDI mode, the analytical sensitivity in UV-MALDI was found to be lower by one order of magnitude, whereas unspecific analyte ion fragmentation as well as adduct formation was found to be more extensive.

Animals↗
PubMed2005

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.

Animals↗
PubMed2005

Time-resolved imaging of the plume dynamics in infrared matrix-assisted laser desorption/ionization with a glycerol matrix.

The dynamics of the expanding material plume after irradiation of a matrix sample with two different infrared (IR) lasers, an Er:YAG laser of ca. 100 ns and an optical parametric oscillator (OPO) laser system of 6 ns pulse duration, were investigated by imaging the plumes with nanosecond time resolution. Both lasers emitted at an identical wavelength of 2.94 microm. Laser exposure parameters were typical for infrared matrix-assisted laser desorption/ionization mass spectrometry (IR-MALDI-MS); glycerol was employed as a liquid matrix to provide a homogeneous sample and reproducible plume formation. A Nd:YAG laser (532 nm; 8 ns) was used as the illumination source and a CMOS camera with a ten-bit dynamic range served for recording of the images. Dark-field as well as scattered light illumination was employed to preferentially image the gaseous and particulate components of the plume, respectively. During the initial phase of its expansion (ca. 1 micros) the plume appears to consist of a continuous cloud of material of varying density. At later times after exposure, individual particles of several micrometers in size dominate the images. For both laser pulse durations material ejection was observed for times as long as 100 micros postexposure. Subtle but distinct differences in the plume dynamics are observed for the two different pulse durations. They are related to a transition between the regimes below and above acoustic confinement. The experimental findings are compared to results obtained in two previous studies by photoacoustic analysis of the desorption process and IR-laser postionization of the plume.

Glycerol↗
PubMed2004

Characterization of whole fibril-forming collagen proteins of types I, III, and V from fetal calf skin by infrared matrix-assisted laser desorption ionization mass spectrometry.

Fibril-forming collagen proteins of the types I, III, and V were extracted from fetal calf skin, purified by differential salt precipitation, and analyzed by infrared matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (IR-MALDI-TOF-MS). Glycerol was used as liquid IR-MALDI matrix. Noncovalently bound triple helices of the types I and V were detected from the NaCl precipitate. After heating at 43 degrees C for 10 min, resulting in the dissociation of the triple helix, the single alpha-chain subunits were detected. For type I, mass spectra acquired from molecular sieve chromatography fractions revealed the presence of further substructures of dimeric type and of supramolecular complexes up to the tetramer. Triple helices of type III, stabilized by covalent disulfide bonds, were detected from the total protein precipitate also after heat treatment. For type III, even hexamer and nonamer structures with molecular weights close to 600 and 900 kDa were recorded. For comparison, ultraviolet (UV-)MALDI-MS measurements with 2,5-dihydroxybenzoic acid as matrix were carried out with some of the samples. Here, only the single alpha-chains were detected with significantly reduced sensitivity.

Animals↗
PubMed2004

Infrared laser post-ionization of large biomolecules from an IR-MALD(I) plume.

A two-infrared laser desorption/ionization method is described. A first laser, which was either an Er:YAG laser or an optical parametric oscillator (OPO), served for ablation/vaporization of small volumes of analyte/matrix sample at fluences below the ion detection threshold for direct matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). A second IR-laser, whose beam intersected the expanding ablation plume at a variable distance and time delay, was used to generate biomolecular ions out of the matrix-assisted laser desorption (MALD) plume. Either one of the two above lasers or an Er:YSGG laser was used for post-ionization. Glycerol was used as IR-MALDI matrix, and mass spectra of peptides, proteins, as well as nucleic acids, some of which in excess of 10(5) u in molecular weight, were recorded with a time-of-flight mass spectrometer. A mass spectrum of cytochrome c from a water ice matrix is also presented. The MALD plume expansion was investigated by varying the position of the post-ionization laser beam above the glycerol sample surface and its delay time relative to the desorption laser. Comparison between the OPO (pulse duration, tau(L) = 6 ns) and the Er:YAG laser (tau(L) approximately 120 ns) as primary excitation laser demonstrates a significant effect of the laser pulse duration on the MALD process.

Angiotensin I↗
PubMed2002

Measurements of mean initial velocities of analyte and matrix ions in infrared matrix-assisted laser desorption ionization mass spectrometry.

The mean initial velocities of analyte ions ranging in molecular weight from 1000 Da to 150 kDa and desorbed with a pulsed Er:YAG laser from various solid-state and liquid IR MALDI matrices were measured along with those of the matrix ions. Experiments with UV MALDI were performed for comparison in addition for a 2,5-dihydroxybenzoic acid preparation. Two different measurement principles were employed, (1) a delayed extraction method, relying on the initial velocity-dependent increase of flight times with delay time between laser and HV ion extraction pulse, and (2) a field-free drift method in which the first region of a two-stage ion source was varied in length and the flight times compared. The two methods yielded somewhat different values for the mean initial ion velocities. Based on a detailed discussion of the measurement principles it is suggested that the actual initial velocities of IR MALDI ions lie between the limits set by the two methods. The influences of the analyte-to-matrix ratio, laser fluence, and laser wavelength on the initial ion velocities were also investigated. Significant differences between the desorption mechanisms for liquid and solid-state matrices were observed.

Journal Article↗
PubMed2002

The role of the laser pulse duration in infrared matrix-assisted laser desorption/ionization mass spectrometry.

The role of the laser pulse duration in matrix-assisted laser desorption/ionization mass spectrometry with infrared lasers (IR-MALDI-MS) emitting in the 3 microm wavelength range has been evaluated. Mass spectrometric performance and characteristics of the IR-MALDI process were examined by comparing a wavelength-tuneable mid-infrared optical parametric oscillator (OPO) laser of 6 ns pulse duration, tuned to wavelengths of 2.79 and 2.94 microm, with an Er:YAG laser (lambda = 2.94 microm) with two pulse durations of 100 and 185 ns, and an Er:YSGG laser (lambda = 2.79 microm) with a pulse duration of 75 ns. Threshold fluences for the desorption of cytochrome C ions were determined as a function of the laser pulse duration for various common IR-MALDI matrices. For the majority of these matrices a reduction in threshold fluence by a factor of 1.2-1.9 was found by going from the 75-100 ns long pulses of the Erbium lasers to the short 6 ns OPO pulse. Within the experimental accuracy threshold fluences were equal for the 100 and the 185 ns pulse duration of the Er:YAG laser. Some pronounced pulse duration effects related to the ion formation from a glycerol matrix were also observed. The effect of the laser pulse length on the duration of ion emission was furthermore investigated.

Angiotensin I↗