Bernhard Spengler

Post-source decay analysis in matrix-assisted laser desorption/ionization mass spectrometry of biomolecules

An introduction to primary structure analysis of biomolecules by matrix-assisted laser desorption/ionization (MALDI) post-source decay (PSD) is given, sketching the principles and applications of the method for scientists in molecular biology, biochemistry and biomedicine. The fundamentals of PSD are described in order to explain the potential and limitations of its applicability. Two examples of peptide sequencing of a completely unknown peptide and of a database-listed peptide are presented and the procedure of (non-automated) amino acid sequence elucidation is described. 1997 by John Wiley & Sons, Ltd.

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.

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 × 100 μm are scanned with minimal increments of 0.25 μm, 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 μm 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.

Peptide and protein identification by matrix-assisted laser desorption ionization (MALDI) and MALDI-post-source decay time-of-flight mass spectrometry

The potential of matrix-assisted laser desorption ionization (MALDI) and MALDI-post-source decay (PSD) time-of-flight mass spectrometry for the characterization of peptides and proteins is discussed. Recent instrumental developments provide for levels of sensitivity and accuracy that make these techniques major analytical tools for proteome analysis. New software developments employing protein database searches have greatly enhanced the fields of application of MALDI-PSD. Peptides and proteins can be easily identified even if only a partial sequence information is determined. Derivatization procedures have been optimized for MALDI-PSD to increase the structural information and to obtain a complete peptide sequence even in critical cases. They are fast, simple and can be performed on target. MALDI-PSD is also a very powerful tool to characterize or elucidate post-translational or chemically induced modifications. In association with database searches, proteins issued from electrophoretic gels can be identified after specific enzymatic cleavages and peptide mapping.

Histology by mass spectrometry: label-free tissue characterization obtained from high-accuracy bioanalytical imaging.

Histological examination of biological and medical specimens has gained its universality and undisputed significance through distinct staining techniques and microscopical evaluation. Discrimination of tissue types after specific staining or labeling is an essential prerequisite for histopathological investigation, for example in accurate diagnosis of cancer. Histochemical staining techniques can only be used in a targeted manner for known compounds, and only a limited number of such targets can be visualized from a given sample at the same time. Another limitation of classical histology lies in the fact that a considerable amount of experience is required and that even well-trained pathologists often interpret histologically stained sections differently. Mass spectrometry (MS), on the other hand, offers complex but objective and reproducible information on biological material. Imaging of biological samples by MS gained interest after development of matrix-assisted laser desorption/ionization (MALDI) as a method to desorb and ionize biomolecules, such as peptides, proteins, glycans, or lipids, with a limit of detection in the attomole range. The first proof-of-principle of imaging by MALDI was presented in 1994, and was followed by numerous applications during the last decade. An extensive overview of instrumental developments and methodological approaches in MS imaging has been published recently. MS imaging allows the distribution of analytes to be investigated and displayed across a sample in a semi-quantitative manner and without the need to predefine or label selected substances prior to analysis. MALDI imaging is typically used with spatial resolutions of between 50 and 200 mm. Increasing the resolution into the lowmicrometer range has been demonstrated, but requires a very low limit of detection of the employed mass spectrometer, as the available amount of material per imaged spot is reduced quadratically with reduction of the spot diameter. Identification of molecules during MS imaging experiments is often limited if mass spectrometers with a rather low mass resolving power and accuracy are used. Additional offline bulk analyses of tissue material are typically used to back up imaging results. Imaging selectivity, that is, mass bin width for allocation to image signals, is typically set to onemass unit. Employing MS imaging for obtaining valid histological information requires a number of improvements: 1. The usable spatial resolution has to be high enough to resolve cellular features. 2. Analytical sensitivity has to be high enough to visualize the majority of interesting substances in high-lateralresolution experiments. 3. Mass resolving power and mass accuracy have to be as high as possible when complex biological samples are under investigation. To unequivocally assign a mass signal to an image and to identify substances by accurate mass, signals have to be stable and correct in detected mass values; that is, mass accuracy should be in the low-ppm range. 4. Image assignment to mass signals has to be both highly selective and flexible. To distinguish neighboring mass signals in biological tissue samples, the coding mass bin width must typically be smaller than 0.1 mass units. 5. To clearly identify imaged substances in complex samples, MS data from fragmentation of precursor ions has to be obtainable directly from individual imaged sample spots. 6. Ambient pressure conditions are often necessary, rather than high-vacuum conditions, for example when working under physiological conditions, imaging volatile substances such as drug metabolites, or using volatile matrices. 7. Sample handling and preparation have to be fast and robust. 8. Results have to be achievable in a reasonable timeframe.

Matrix vapor deposition/recrystallization and dedicated spray preparation for high‐resolution scanning microprobe matrix‐assisted laser desorption/ionization imaging mass spectrometry (SMALDI‐MS) of tissue and single cells

Matrix preparation techniques such as air spraying or vapor deposition were investigated with respect to lateral migration, integration of analyte into matrix crystals and achievable lateral resolution for the purpose of high-resolution biological imaging. The accessible mass range was found to be beyond 5000 u with sufficient analytical sensitivity. Gas-assisted spraying methods (using oxygen-free gases) provide a good compromise between crystal integration of analyte and analyte migration within the sample. Controlling preparational parameters with this method, however, is difficult. Separation of the preparation procedure into two steps, instead, leads to an improved control of migration and incorporation. The first step is a dry vapor deposition of matrix onto the investigated sample. In a second step, incorporation of analyte into the matrix crystal is enhanced by a controlled recrystallization of matrix in a saturated water atmosphere. With this latter method an effective analytical resolution of 2 microm in the x and y direction was achieved for scanning microprobe matrix-assisted laser desorption/ionization imaging mass spectrometry (SMALDI-MS). Cultured A-498 cells of human renal carcinoma were successfully investigated by high-resolution MALDI imaging using the new preparation techniques.

Single Cell Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

Application of mass spectrometry imaging (MS imaging) analysis to single cells was so far restricted either by spatial resolution in the case of matrix-assisted laser desorption/ionization (MALDI) or by mass resolution/mass range in the case of secondary ion mass spectrometry (SIMS). In this study we demonstrate for the first time the combination of high spatial resolution (7 μm pixel), high mass accuracy (<3 ppm rms), and high mass resolution (R = 100,000 at m/z = 200) in the same MS imaging measurement of single cells. HeLa cells were grown directly on indium tin oxide (ITO) coated glass slides. A dedicated sample preparation protocol was developed including fixation with glutaraldehyde and matrix coating with a pneumatic spraying device. Mass spectrometry imaging measurements with 7 μm pixel size were performed with a high resolution atmospheric-pressure matrix-assisted laser desorption/ionization (AP-MALDI) imaging source attached to an Exactive Orbitrap mass spectrometer. Selected ion images were generated with a bin width of Δm/z = ±0.005. Selected ion images and optical fluorescence images of HeLa cells showed excellent correlation. Examples demonstrate that a lower mass resolution and a lower spatial resolution would result in a significant loss of information. High mass accuracy measurements of better than 3 ppm (root-mean-square) under imaging conditions provide confident identification of imaged compounds. Numerous compounds including small metabolites such as adenine, guanine, and cholesterol as well as different lipid classes such as phosphatidylcholine, sphingomyelin, diglycerides, and triglycerides were detected and identified based on a mass spectrum acquired from an individual spot of 7 μm in diameter. These measurements provide molecularly specific images of larger metabolites (phospholipids) in native single cells. The developed method can be used for a wide range of detailed investigations of metabolic changes in single cells.

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.

Controlling the enzymatic activity of a restriction enzyme by light

For many applications it would be desirable to be able to control the activity of proteins by using an external signal. In the present study, we have explored the possibility of modulating the activity of a restriction enzyme with light. By cross-linking two suitably located cysteine residues with a bifunctional azobenzene derivative, which can adopt a cis- or trans-configuration when illuminated by UV or blue light, respectively, enzymatic activity can be controlled in a reversible manner. To determine which residues when cross-linked show the largest “photoswitch effect,” i.e., difference in activity when illuminated with UV vs. blue light, > 30 variants of a single-chain version of the restriction endonuclease PvuII were produced, modified with azobenzene, and tested for DNA cleavage activity. In general, introducing single cross-links in the enzyme leads to only small effects, whereas with multiple cross-links and additional mutations larger effects are observed. Some of the modified variants, which carry the cross-links close to the catalytic center, can be modulated in their DNA cleavage activity by a factor of up to 16 by illumination with UV (azobenzene in cis) and blue light (azobenzene in trans), respectively. The change in activity is achieved in seconds, is fully reversible, and, in the case analyzed, is due to a change in Vmax rather than Km.

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.