Dieter Kirsch

Sequenching of peptides in a time-of-flight mass spectrometer: evaluation of postsource decay following matrix-assisted laser desorption ionisation (MALDI)

Abstract In matrix assisted laser desorption ionisation (MALDI) a large fraction of analyte ions undergo postsource decay (PSD) during flight in the field free drift path. By means of a modified two-state reflectron, daughter ion time-of-flight spectra of medium sized linear peptides (up to 2800 u) were recorded containing full sequence information. Precision, accuracy and mass resolution of fragment ions were almost as good as obtained in high energy CAD studies performed in four-sector instruments. Instrumental sensitivity was better at least one order of magnitude. In reflectron time-of-flight mass spectrometry (RETOF-MS) the cleavage pattern of PSD products is different from that obtained by high energy and low energy CAD. In our instrument, conditions which were energetically comparable to high energy and low energy CAD could easily and comparatively be studied in the same experiment by varying instrumental parameters. Activation mechanisms of PSD were found to be largely determined by collisional events (ion/neutral) induced by the acceleration field during early plume expansion. Future potentials of PSD analysis after MALDI are discussed.

PRION RODS CONTAIN SMALL AMOUNTS OF TWO HOST SPHINGOLIPIDS AS REVEALED BY THIN-LAYER CHROMATOGRAPHY AND MASS SPECTROMETRY

Sphingolipids were detected in prions, the agents of transmissible spongiform encephalopathies. The analysis was carried out on highly purified, infectious prion rods, which are composed mainly of insoluble aggregates of the N-terminally truncated prion protein, so-called PrP 27-30. Lipid classes were quantified by high performance thin-layer chromatography with a detection limit of 25-50 ng per lipid class. Matrix-assisted laser desorption/ionization mass spectrometry was applied for the first time to lipid analysis in complex biological samples. A newly developed preparation technique improved the sensitivity to 1-20 pg per molecular species. Only the sphingolipids, galactosylceramide and sphingomyelin, were consistently observed in chloroform/methanol (2:1 v/v) extracts of prion rods. The molar ratio of PrP to the sphingolipids was between 2:1 and 40:1, depending on the purity of the prion preparation. The same lipids were also present in the low density fraction of a gradient centrifugation of prion-rods after sonication in 0.2% SDS. From the two alternatives, that the sphingolipids are either required for prion function or are relics from the cellular location of PrP in caveolae, the second alternative appears more plausible since the preparation of highest specific infectivity contained the lowest amount of sphingolipids.

Post‐source Decay and Delayed Extraction in Matrix‐assisted Laser Desorption/Ionization‐Reflectron Time‐of‐Flight Mass Spectrometry. Are There Trade‐offs?

By the incorporation of delayed extraction (DE) into matrix-assisted laser desorption/ionization time-of-flight mass spectrometry a dramatic improvement of performance with respect to sensitivity, mass resolution and mass accuracy of precursor ions up to approximately 10 kDa has been achieved. Since DE reduces collisional in-source activation to a large extent, the rate of subsequent metastable decay is considerably reduced. Results are presented which demonstrate that under DE the loss of total post-source decay (PSD) fragment ion yield can be as large as one order of magnitude but that, in terms of sensitivity, part of this loss is balanced by a better S/N ratio which results from a significantly improved mass resolution of the PSD fragment ions (M/delta M up to 1800 compared with M/delta M = 200-500 under prompt extraction). While this compensatory effect is true for the middle to high mass range of PSD fragment ions, it gradually vanishes towards the low mass end of the PSD mass scale where, in the case of linear peptides some important information (immonium ions) is lost. It appears, however, that in the majority of practical PSD work, DE improves the qualty of the PSD spectra and that high energy collisional post-source activation can compensate for the occasional loss of analytical information.

A high-resolution scanning microprobe matrix-assisted laser desorption/ionization ion source for imaging analysis on an ion trap/Fourier transform ion cyclotron resonance mass spectrometer

A new scanning microprobe matrix-assisted laser desorption/ionization (SMALDI) ion source for high spatial resolution has been developed for linear ion trap and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). The source is fully compatible with commercial ion trap flanges (such as the LTQ series, Thermo Fisher Scientific). The source is designed for atmospheric pressure (AP) operation but is also suitable for mid-pressure operation. The AP mode is especially useful for investigating volatile compounds. The source can be interchanged with other ion sources within a minute when operated in the AP mode. Combining high-lateral resolution MALDI imaging with high mass resolution and high mass accuracy mass spectrometry, available in the FT-ICR mode, provides a new quality of analytical information, e.g. from biological samples. First results obtained with the new ion source demonstrate a maximum lateral resolution of 0.6 by 0.5 microm. Depending on the limit of detection of the chosen mass analyzer, however, the size of the focus had to be enlarged to a diameter of up to 8 microm in the FT-ICR mode, in order to create enough ions for detection. Mass spectra acquired for analytical imaging were obtained from single laser pulses per pixel in all the experiments. This mode allows us to investigate biological thin sections with desorption focus diameters in the micrometer range, known to cause complete evaporation of material under the laser focus with a very limited number of laser pulses. As a first example, peptide samples deposited in microstructures were investigated with the new setup. A high quality and validity of the acquired images were obtained in the ion trap mode due to the low limit of detection. High mass resolution and accuracy but poorer image quality were obtained in the ICR mode due to the lower detection sensitivity of the ICR detector.

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.

On the formation of initial ion velocities in matrix-assisted laser desorption ionization: Virtual desorption time as an additional parameter describing ion ejection dynamics

Abstract Measurements of initial ion velocities prior to ion acceleration were performed using a special instrumental setup with a variable length of a first field-free stage in a two-stage MALDI ion source. Experimental results showed that drift times through the field-free stage cannot be extrapolated to zero when reducing the length of this stage, but indicate a condition-specific temporal offset. This offset can mathematically be interpreted as a delayed start of ions and is therefore called “virtual desorption time”. Mechanistically these virtual desorption times have to be interpreted as being the result of acceleration processes of analyte ions in a molecular jet, leading to various dependencies of analyte ion velocities and virtual desorption times on the chosen experimental parameters, rather than being the result of a delayed desorption of molecular ions or clusters from the surface. Formation of analyte ions by cluster decay is in accordance with the reported observations, as is formation of analyte ions by charge exchange in the gas phase. In both cases, however, the presence of an efficient molecular jet is suggested by these experimental results, leading to a subsequent acceleration of entrained molecular ions after detachment of molecular entities from the solid phase.

Evaluation of the Photodegradation of Crystal Violet upon Light Exposure by Mass Spectrometric and Spectroscopic Methods

Abstract:  Crystal violet is a very common dye in ballpoint ink. Recent research suggests that the degradation of triarylmethane dyes gives an indication of the age of a ballpoint pen entry on a document. The main problem for the quantitative evaluation of the degradation is that it is highly dependent on the exposure to light. Moreover additional factors, such as additives and substrate play an important role in this process. The aim of this work is to compare the degradation pathways of the pure dye in water and ethanol upon exposure to xenon light by UV/VIS spectrophotometry and laser desorption ionization. Significant differences have been observed in the products and the kinetics of the degradation. N‐demethylation, an expected decomposition process, was found to take place only in aqueous solution and kinetics calculations showed that the degradation occurred 2.5 times faster in ethanol compared to water. The degradation of crystal violet in inks from four ballpoint pens on paper was also studied for entries made over 2–3 years. It was observed that degradation reactions were quenched by the presence of another dye due to competitive absorption. It was also observed that the thickness of a stroke (concentration of ink) influenced the degradation process. In the absence of light only one ballpoint pen showed slight degradation. A better understanding of the influence of the paper, ink composition, and storage conditions is necessary to interpret correctly the age of an ink based on the degradation of dyes.

Dimeric Cyclodextrin Carriers with High Binding Affinity to Porphyrinoid Photosensitizers

The aim of our investigation was to develop carrier systems for an application of inert drugs in photodynamic cancer therapy. β-Cyclodextrin dimers linked at their primary and secondary faces by spacers of varying lengths were synthesized as carrier systems. The binding constants of the inclusion complexes of these cyclodextrin dimers and porphyrinoid photosensitizers were determined by competitive spectrofluorometry. Particularly the secondary face linked dimers exhibited extremely high binding constants with values of 106-107 L/mol. Theoretical studies were carried out on these inclusion complexes to confirm the influence of spacer length and connecting side on complex stability.

Ink Dating Using Thermal Desorption and Gas Chromatography/Mass Spectrometry: Comparison of Results Obtained in Two Laboratories

An ink dating method based on solvent analysis was recently developed using thermal desorption followed by gas chromatography/mass spectrometry (GC/MS) and is currently implemented in several forensic laboratories. The main aims of this work were to implement this method in a new laboratory to evaluate whether results were comparable at three levels: (i) validation criteria, (ii) aging curves, and (iii) results interpretation. While the results were indeed comparable in terms of validation, the method proved to be very sensitive to maintenances. Moreover, the aging curves were influenced by ink composition, as well as storage conditions (particularly when the samples were not stored in “normal” room conditions). Finally, as current interpretation models showed limitations, an alternative model based on slope calculation was proposed. However, in the future, a probabilistic approach may represent a better solution to deal with ink sample inhomogeneity.

Synthesis of β-cyclodextrin dimers as carrier systems for photodynamic therapy of cancer

The aim of our investigation was the development of carrier systems for an application of inert drugs in polyphasic photodynamic tumor therapy. As carrier systems, β-cyclodextrin dimers linked at their primary and secondary faces by spacers of varying lengths were synthesized. Cyclodextrins are known to form stable inclusion complexes with porphyrinoid photosensitizers. The influence of spacer length on the β-cyclodextrin dimer inclusion complexes with porphyrinoid photosensitizers was studied.