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Biomedical subjects

Bernhard Spengler

Publications and source records attributed to Bernhard Spengler.

11 recordsLinked to original sources
PubMed2007

Differentiation of blue ballpoint pen inks by laser desorption ionization mass spectrometry and high-performance thin-layer chromatography.

The differentiation of inks on a questioned document can highlight a fraudulent insertion and is usually carried out by optical comparison and thin-layer chromatography (TLC). Laser desorption ionization mass spectrometry (LDI-MS) may also be used for the analysis of dyes from ink. This analytical technique was compared with a standard method of high-performance TLC (HPTLC) according to their capacity to differentiate blue ballpoint inks. Ink entries on paper from 31 blue ballpoint pens have been analyzed and their dye ink formulations compared. The pens were classified into 26 classes by LDI-MS against 18 for HPTLC. LDI-MS proved to be a more powerful method for differentiating ink formulations because it provides information about dye structures (molecular weights) and relative quantification of dye classes (peak areas). Sample preparation was minimal and analysis time was short in contrast to the more complex extraction, application, and development steps of the HPTLC method. However, only basic dyes and pigments were identified using positive mode LDI-MS, while HPTLC did yield additional information about acid dyes.

Journal Article↗
PubMed2006

A GC/MS study of the drying of ballpoint pen ink on paper.

The determination of the age of an ink entry from a questioned document is often an essential problem and a controversial issue in forensic sciences. Therefore, it is important to understand the aging process of the different components found in ink. The aim of this work was to study the drying process of ballpoint ink, characterised by the disappearance of volatile solvents from the ink entry. Phenoxyethanol is of particularly high interest as it is found in more than 80% of the blue ballpoint pens at different concentrations. Liquid extraction followed by splitless gas chromatography/mass spectrometry in the selected ion mode was used to measure the quantitative decrease of solvents from ink entries made with a blue Parker ballpoint pen. Quantities of ethoxyethoxyethanol, dipropylene glycol, phenoxyethanol and phenoxyethoxyethanol were studied in ink entries up to 1.5 years old, thus allowing to calculate aging curves for this particular pen. The low quantities of solvents (in the microgram range for a 1 cm ballpoint entry) were found to decrease quickly after deposition of the ink on paper through the competitive processes of evaporation and diffusion. Losses of up to 75% of solvents were observed after a few seconds. The amount of ethoxyethanol stopped decreasing after about 10 days (quantities reached the nanogram range for a 1 cm ballpoint entry), while the aging curves of dipropylene glycol, phenoxyethanol and phenoxyethoxyethanol level off considerably after 2 weeks. It was observed that ethoxyethanol, dipropylene glycol and phenoxyethanol can also migrate from one sheet of paper to another if placed close enough (e.g. in a book or a stack of papers), therefore contamination from fresh ink strokes from other paper sheets has to be taken into account for those solvents. In this paper we demonstrate that differentiation between fresh ink (<2 weeks) and older inks is possible under laboratory storage conditions. For real cases samples, more parameters have to be studied and other possible pathways have to be considered.

Forensic Sciences↗
PubMed2006

Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry.

To investigate protein-protein interaction sites in the DNA mismatch repair system we developed a crosslinking/mass spectrometry technique employing a commercially available trifunctional crosslinker with a thiol-specific methanethiosulfonate group, a photoactivatable benzophenone moiety and a biotin affinity tag. The XACM approach combines photocrosslinking (X), in-solution digestion of the crosslinked mixtures, affinity purification via the biotin handle (A), chemical coding of the crosslinked products (C) followed by MALDI-TOF mass spectrometry (M). We illustrate the feasibility of the method using a single-cysteine variant of the homodimeric DNA mismatch repair protein MutL. Moreover, we successfully applied this method to identify the photocrosslink formed between the single-cysteine MutH variant A223C, labeled with the trifunctional crosslinker in the C-terminal helix and its activator protein MutL. The identified crosslinked MutL-peptide maps to a conserved surface patch of the MutL C-terminal dimerization domain. These observations are substantiated by additional mutational and chemical crosslinking studies. Our results shed light on the potential structures of the MutL holoenzyme and the MutH-MutL-DNA complex.

Adenosine Triphosphatases↗
PubMed2006

Photofading of ballpoint dyes studied on paper by LDI and MALDI MS.

The determination of the age of an ink entry from a questioned document is often a major problem and a controversial issue in forensic sciences. Therefore, it is important to understand the aging process of the different components found in ink. The aim of this work is to characterize the degradation processes of methyl violet and ethyl violet, two typical ballpoint dyes by using laser desorption/ionization (LDI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), and to evaluate the possible application of the method to forensic examination of documents. The mass spectrometric methods were first tested and were found to be adequate for the purpose of this work. Moreover, it is possible to analyze the dye from a stroke directly from the paper (LDI-MS), so the sample preparation is minimized. The degradation of the dyes methyl violet and ethyl violet in strokes from a ballpoint pen was studied under laboratory conditions influenced by different factors such as light, wavelength of light, heat, and humidity. Then, strokes from the same ballpoint were aged naturally in the dark or under the influence of light over one year and then analyzed. The results show that the degradation of these dyes strongly depends on light fluence. Humidity also increases degradation, which can be explained by the basicity of the paper. The influence of heat on the degradation process was found to be rather weak. It was also observed that the dyes from the ink strokes did not show significant degradation after one year of storage in the dark. In conclusion, the storage conditions of a questioned document and the initial composition of the dyes in the ink have to be known for correct interpretation of the age of an ink entry. Measurements over longer periods of time are necessary to follow the degradation of dyes exempt from light exposure. LDI was found adequate and very useful for the analysis of ballpoint dyes directly from paper without further pretreatment.

Coloring Agents↗
PubMed2006

Effective solvation of alkaline earth ions by proline-rich proteolytic peptides of galectin-3 upon electrospray ionisation.

In an analysis of a combined chymotrypsin/AspN digest of galectin-3 by positive ion nano-electrospray ionisation mass spectrometry (nanoESI-MS) several peptides were observed which showed metal adduct ions as their most abundant ion signals. The most prominent adduct ions were observed at m/z values corresponding to [M+40]2+, [M+41]3+, and [M+42]4+ ions. Detailed investigation of the [M+40]2+ ion of the peptide GAPAGPLIVPY showed that it was not, as originally expected, a [M+H+39K]2+ adduct ion but had the composition [M+40Ca]2+. This was verified by several approaches: (i) nanoESI-MS/MS of the [M+Ca]2+ adduct ions resulted in the virtually exclusive formation of doubly charged fragment ions; (ii) mass determination by quadrupole time-of-flight (QTOF)-MS provided a preliminary identification; and (iii) accurate mass measurement using nanoESI Fourier transform ion cyclotron resonance (FTICR)-MS at a mass resolving power of 500 000 allowed the specific detection and identification of the isobaric ion pairs [M+40Ca]2+/[M+H+39K]2+ and [M+24Mg]2+/[M+H+23Na]2+. All peptides in the chymotryptic galectin-3 digest without a basic residue (K or R) showed addition of calcium as the most prominent ionisation principle. A further common feature of these nonbasic peptides was the presence of several proline residues, which is assumed to be a factor promoting the intense addition of calcium. It was observed that the common trace levels of sodium and calcium in analytical grade solvents (about 1-10 microM) are sufficient to generate the [M+H+23Na]2+ and [M+40Ca]2+ ions as the most prominent species of the peptide GAPAGPLIVPY. We conclude that the sequence motifs P-XX-P and P-XXX-P favour the solvation of alkaline earth ions in ESI-MS. In view of the successful detection of physiological Ca/protein interactions by ESI-MS, this finding may point to a solvation of Ca2+ by galectin in solution. The findings open new routes of research in the study of metal/protein and metal/peptide interactions

Amino Acid Sequence↗
PubMed2005

Initial velocity distributions of ions generated by in-flight laser desorption/ionization of individual polystyrene latex microparticles as studied by the delayed ion extraction method.

The delayed ion extraction method has been used to study characteristics of the initial velocity distributions of positive and negative ions produced simultaneously by laser desorption/ionization (LDI) from non-impacted single aerosol polymeric particles, using a bipolar time-of-flight (TOF) instrument (LAMPAS 2). Due to the geometry of the setup and the characteristics of the ablation process, only the projections of the velocities on the axis of the mass spectrometer can be directly studied. Additionally, since the mean initial velocity under these conditions should be close to zero, it was necessary to extend the method by taking into account higher order contributions of the velocity distribution. Theoretical expressions for these higher order terms are presented and discussed. The bipolar characteristics of the instrument permit evaluation and treatment of a possible instrumental artifact caused by small inclinations of the ionizing laser with respect to the ideal incidence direction. Results of a number of experiments are presented and discussed in relation to the theoretical expressions presented, and to possible ablation scenarios. Evidence pointing out that, under our experimental conditions, only partial ablation of the latex particles occurs was obtained. The variance of the distribution of the projection of the initial velocities can be directly estimated from these results. By assuming that the total initial velocities of the ions are developed completely according to a single-temperature adiabatic expansion mechanism, temperatures of approximately 50 K/Da can be assigned to the ion clouds from the variance estimations. If a two-temperature model is used, a radial temperature of about 100 K/Da results. These values are in reasonable agreement with results for polymer ablation from the literature.

Ions↗
PubMed2004

Mapping protein-protein interactions between MutL and MutH by cross-linking.

Strand discrimination in Escherichia coli DNA mismatch repair requires the activation of the endonuclease MutH by MutL. There is evidence that MutH binds to the N-terminal domain of MutL in an ATP-dependent manner; however, the interaction sites and the molecular mechanism of MutH activation have not yet been determined. We used a combination of site-directed mutagenesis and site-specific cross-linking to identify protein interaction sites between the proteins MutH and MutL. Unique cysteine residues were introduced in cysteine-free variants of MutH and MutL. The introduced cysteines were modified with the cross-linking reagent 4-maleimidobenzophenone. Photoactivation resulted in cross-links verified by mass spectrometry of some of the single cysteine variants to their respective Cys-free partner proteins. Moreover, we mapped the site of interaction by cross-linking different combinations of single cysteine MutH and MutL variants with thiol-specific homobifunctional cross-linkers of varying length. These results were used to model the MutH.MutL complex and to explain the ATP dependence of this interaction.

Adenosine Triphosphatases↗
PubMed2004

De novo sequencing, peptide composition analysis, and composition-based sequencing: a new strategy employing accurate mass determination by fourier transform ion cyclotron resonance mass spectrometry.

A new strategy is described for the determination of amino acid sequences of unknown peptides. Different from the well-known but often inefficient de novo sequencing approach, the new method is based on a two-step process. In the first step the amino acid composition of an unknown peptide is determined on the basis of accurate mass values of the peptide precursor ion and a small number of accurate fragment ion mass values, and, as in de novo sequencing, without employing protein database information or other pre-information. In the second step the sequence of the found amino acids of the peptide is determined by scoring the agreement between expected and observed fragment ion signals of the permuted sequences. It was found that the new approach is highly efficient if accurate mass values are available and that it easily outstrips common approaches of de novo sequencing being based on lower accuracies and detailed knowledge of fragmentation behavior. Simple permutation and calculation of all possible amino acid sequences, however, is only efficient if the composition is known or if possible compositions are at least reduced to a small list. The latter requires the highest possible instrumental mass accuracy, which is currently provided only by fourier transform ion cyclotron resonance mass spectrometry. The connection between mass accuracy and peptide composition variability is described and an example of peptide compositioning and composition-based sequencing is presented.

Amino Acid Sequence↗
PubMed2002

Scanning microprobe matrix-assisted laser desorption ionization (SMALDI) mass spectrometry: instrumentation for sub-micrometer resolved LDI and MALDI surface analysis.

A new instrument and method is described for laterally resolved mass spectrometric surface analysis. Fields of application are in both the life sciences and the material sciences. The instrument provides for imaging of the distribution of selected sample components from natural and artificial surfaces. Samples are either analyzed by laser desorption ionization (LDI) time-of-flight mass spectrometry or, after preparation with a suitable matrix, by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Areas of 100 x 100 microm are scanned with minimal increments of 0.25 microm, and between 10,000 and 160,000 mass spectra are acquired per image within 3 to 50 min (scan rate up to 50 pixels per s). The effective lateral resolution is in the range of 0.6 to 1.5 microm depending on sample properties, preparation methods and laser wavelength. Optical investigation of the same sample area by UV confocal scanning laser microscopy was found to be very attractive in combination with scanning MALDI mass analysis because pixel-identical images can be created with both techniques providing for a strong increase in analytical information. This article describes the method and instrumentation, including first applicational examples in elemental analysis, imaging of pine tree roots, and investigation of MALDI sample morphology in biomolecular analysis.

Algorithms↗