Raimund Kaufmann

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.

Simultaneous Detection of Positive and Negative Ions From Single Airborne Particles by Real-time Laser Mass Spectrometry

The instrumental setup used for simultaneous bipolar ion detection in on-line single-particle analysis by laser mass spectrometry is described. Particle size calibration has been performed using aerodynamic time-of-flight techniques. Mass spectra of several hundred ambient particles with aerodynamic diameters of 0.8 μm have been evaluated for statistical classification (Principal Components Analysis [PCA]) in order to identify main chemical components of the prominent particle classes.

Dynamical parameters of ion ejection and ion formation in matrix-assisted laser desorption/ionization

Molecular ejection and ionization processes in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry are described. Angular resolved initial velocity distributions of matrix and analyte ions, obtained both from MALDI samples prepared normally and from peptide-doped single crystals of dihydroxybenzoic acid, are presented as ion density plots. The ejection distributions have been found to be bimodal, consisting of a slow, less forward peaked fraction and a fast fraction, which is directed more towards the surface normal. The ion plots show a significant quenching of matrix ions in areas of high peptide ion density. This effect is interpreted as the result of ion–molecule reactions in the gas phase leading to proton transfer from matrix ions to analyte molecules and is described by a simple kinetic overlap model. Additional effects of solid phase absorption, electronic excitation life times and fluorescence yield are discussed.

Peptide sequencing of charged derivatives by postsource decay MALDI mass spectrometry

Abstract Derivatization procedures for peptides are described that can be performed with sub-picomolar amounts of sample and that are able to direct the formation of fragment ions in Postsource Decay (PSD) MALDI mass spectrometry. Location of a fixed charge (a quarternary ammonium ion) at the N-terminus of a peptide and modification of internal arginine residues (deletion of strong basicity) leads to a full controllability of fragment ion formation resulting in mostly complete series of N-terminal fragment ions. The method appears to be favorably applicable to sequence analysis of unknown peptides, since in most cases the amino acid sequence can directly be read from the spectrum.

Isolation and structural elucidation of two pyrokinins from the retrocerebral complex of the American cockroach.

By monitoring the contractile activity of the hyperneural muscle of the American cockroach in vitro two peptides were isolated from the retrocerebral complex of the American cockroach. Three purification steps using reversed-phase high performance liquid chromatography on C-18 columns containing trifluoroacetic acid or heptafluorobutyric acid as organic modifiers were sufficient to achieve homogeneous peptide preparations. The structures of both peptides were elucidated by a combination of Edman degradation and mass spectrometry which yielded the following structures: His-Thr-Ala-Gly Phe-Ile-Pro-Arg-Leu-NH2 (Pea-PK-1) and Ser-Pro-Pro-Phe-Ala-Pro-Arg-Leu-NH2 (Pea-PK-2). The C-terminal sequence Phe-X-Pro-Arg-Leu-NH2 characterized the peptides as members of the insect pyrokinin family. The synthetic peptides were shown to have the same retention times as the natural peptides. The occurrence of both peptides in the retrocerebral complex suggests a physiological role as neurohormones. The effects of the synthetic pyrokinis were clearly distinguishable in their actions on the hyperneural muscle. Regarding the threshold concentrations, Pea-PK-2 was only 0.3% as active as Pea-PK-1.

Drug-induced zeta potential changes in liposomes studied by laser doppler spectroscopy

The technique of laser Doppler spectroscopy is used to measure the electrophoretic mobility of liposomes under the influence of one beta-blocking agent and three local anesthetics. All four drugs decrease the mobility (i.e., the zeta potential) of negatively charged phospholipids (soybean lipids, phosphatidylserine and cardiolipin). The mobility of electrostatically neutral pure phosphatidylcholine (zero mobility under control conditions at pH 7 and 4) is increased linearly with the logarithm of drug concentration, indicating binding and incorporation of positively charged drug molecules. The sequence of strength of activity, measured by zeta-potential changes, corresponds to that found in biological tissues: propranolol greater than tetracaine greater than lidocaine greater than procaine. For purely negatively charged lipids (phosphatidylserine, cardiolipin) the activity of the drug is higher at acidic pH, (pH 4), while for electrostatically neutral (phosphatidylcholine) or partly neutral (soybean) lipid liposomes drug activity is about the same at pH 9, 7 and 4. A Hill plot of the data reveals noncooperative drug binding. From the line width of the scattering power spectrum the mean particle radius and the average interparticle distance in the samples are determined.

Laser Doppler spectroscopy as applied to electrophoresis in protein solutions

Abstract The application of Laser Doppler spectroscopy (LDS) to the electrophoretic migration of macromolecules in solution by heterodyne light beating technique, previously developed by Ware and Flygare, has been improved by the design of a new microelectrophoresis cell and a high resolution in the frequency power spectrum. A 1024-channel correlator was used in combination with a software-controlled Fast Fourier transformation (FFT). This technique has been applied to single protein solution, bovine serum albumin (BSA), and to multicomponent systems, in particular to human blood serum. In comparison to normal free electrophoresis, LDS is more convenient and reveals more information in a much shorter period of time.

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.