Biomedical subjects

Ron M A Heeren

Publications and source records attributed to Ron M A Heeren.

At least 19 recordsLinked to original sources

PubMed2006

Imaging of peptides in the rat brain using MALDI-FTICR mass spectrometry.

Analytical methods are pursued to measure the identity and location of biomolecules down to the subcellular (microm) level. Available mass spectrometric imaging methods either compromise localization accuracy or identification accuracy in their analysis of surface biomolecules. In this study, imaging FTICR-MS is applied for the spatially resolved mass analysis of rat brain tissue with the aim to optimize protein identification by the high mass accuracy and online MS/MS capabilities of the technique. Mass accuracies up to 6 ppm were obtained in the direct MALDI-analysis of the tissue together with a spatial resolution of 200 microm. The spatial distributions of biomolecules differing in mass by less than 0.1 Da could be resolved, and are shown to differ significantly. Online MS/MS analysis of selected ions was demonstrated. A comparison of the FTICR-MS imaging results with stigmatic TOF imaging on the same sample is presented. To reduce the extended measuring times involved, it is recommended to restrict the FTICR-MS analyses to areas of interest as can be preselected by other, faster imaging methods.

Animals↗

PubMed2006

Electron capture dissociation as structural probe for noncovalent gas-phase protein assemblies.

Electron capture dissociation (ECD) of proteins in Fourier transform ion cyclotron resonance mass spectrometry usually leads to charge reduction and backbone-bond cleavage, thereby mostly retaining labile, intramolecular noncovalent interactions. In this report, we evaluate ECD of the 84-kDa noncovalent heptameric gp31 complex and compare this with sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD) of the same protein. Unexpectedly, the 21+ charge state of the gp31 oligomer exhibits a main ECD pathway resulting in a hexamer and monomer, disrupting labile, intermolecular noncovalent bonds and leaving the backbone intact. Unexpectedly, the charge separation over the two products is highly proportional to molecular weight. This indicates that a major charge redistribution over the subunits of the complex does not take place during ECD, in contrast to the behavior observed when using SORI-CAD. We speculate that the ejected monomer retains more of its original structure in ECD, when compared to SORI-CAD. ECD of lower charge states of gp31 does not lead to dissociation of noncovalent bonds. We hypothesize that the initial gas-phase structure of the 21+ charge state is significantly different from the lower charge states. These structural differences result in the different reaction pathways when using ECD.

Chaperonins↗

PubMed2006

A database application for pre-processing, storage and comparison of mass spectra derived from patients and controls.

BACKGROUND: Statistical comparison of peptide profiles in biomarker discovery requires fast, user-friendly software for high throughput data analysis. Important features are flexibility in changing input variables and statistical analysis of peptides that are differentially expressed between patient and control groups. In addition, integration the mass spectrometry data with the results of other experiments, such as microarray analysis, and information from other databases requires a central storage of the profile matrix, where protein id's can be added to peptide masses of interest. RESULTS: A new database application is presented, to detect and identify significantly differentially expressed peptides in peptide profiles obtained from body fluids of patient and control groups. The presented modular software is capable of central storage of mass spectra and results in fast analysis. The software architecture consists of 4 pillars, 1) a Graphical User Interface written in Java, 2) a MySQL database, which contains all metadata, such as experiment numbers and sample codes, 3) a FTP (File Transport Protocol) server to store all raw mass spectrometry files and processed data, and 4) the software package R, which is used for modular statistical calculations, such as the Wilcoxon-Mann-Whitney rank sum test. Statistic analysis by the Wilcoxon-Mann-Whitney test in R demonstrates that peptide-profiles of two patient groups 1) breast cancer patients with leptomeningeal metastases and 2) prostate cancer patients in end stage disease can be distinguished from those of control groups. CONCLUSION: The database application is capable to distinguish patient Matrix Assisted Laser Desorption Ionization (MALDI-TOF) peptide profiles from control groups using large size datasets. The modular architecture of the application makes it possible to adapt the application to handle also large sized data from MS/MS- and Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometry experiments. It is expected that the higher resolution and mass accuracy of the FT-ICR mass spectrometry prevents the clustering of peaks of different peptides and allows the identification of differentially expressed proteins from the peptide profiles.

Algorithms↗

PubMed2006

Higher sensitivity secondary ion mass spectrometry of biological molecules for high resolution, chemically specific imaging.

To expand the role of high spatial resolution secondary ion mass spectrometry (SIMS) in biological studies, numerous developments have been reported in recent years for enhancing the molecular ion yield of high mass molecules. These include both surface modification, including matrix-enhanced SIMS and metal-assisted SIMS, and polyatomic primary ions. Using rat brain tissue sections and a bismuth primary ion gun able to produce atomic and polyatomic primary ions, we report here how the sensitivity enhancements provided by these developments are additive. Combined surface modification and polyatomic primary ions provided approximately 15.8 times more signal than using atomic primary ions on the raw sample, whereas surface modification and polyatomic primary ions yield approximately 3.8 and approximately 8.4 times more signal. This higher sensitivity is used to generate chemically specific images of higher mass biomolecules using a single molecular ion peak.

Animals↗

PubMed2006

Tandem mass spectrometry of intact GroEL-substrate complexes reveals substrate-specific conformational changes in the trans ring.

It has been suggested that the bacterial GroEL chaperonin accommodates only one substrate at any given time, due to conformational changes to both the cis and trans ring that are induced upon substrate binding. Using electrospray ionization mass spectrometry, we show that indeed GroEL binds only one molecule of the model substrate Rubisco. In contrast, the capsid protein of bacteriophage T4, a natural GroEL substrate, can occupy both rings simultaneously. As these substrates are of similar size, the data indicate that each substrate induces distinct conformational changes in the GroEL chaperonin. The distinctive binding behavior of Rubisco and the capsid protein was further investigated using tandem mass spectrometry on the intact 800-914 kDa GroEL-substrate complexes. Our data suggest that even in the gas phase the substrates remain bound inside the GroEL cavity. The analysis revealed further that binding of Rubisco to the GroEL oligomer stabilizes the chaperonin complex significantly, whereas binding of one capsid protein did not have the same effect. However, addition of a second capsid protein molecule to GroEL resulted in a similar stabilizing effect to that obtained after the binding of a single Rubisco. On the basis of the stoichiometry of the GroEL chaperonin-substrate complex and the dissociation behavior of the two different substrates, we hypothesize that the binding of a single capsid polypeptide does not induce significant conformational changes in the GroEL trans ring, and hence the unoccupied GroEL ring remains accessible for a second capsid molecule.

Bacteriophage T4↗

PubMed2006

Gold-enhanced biomolecular surface imaging of cells and tissue by SIMS and MALDI mass spectrometry.

Surface metallization by plasma coating enhances desorption/ionization of membrane components such as lipids and sterols in imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) of tissues and cells. High-resolution images of cholesterol and other membrane components were obtained for neuroblastoma cells and revealed subcellular details (resolving power 1.5 mum). Alternatively, in matrix-enhanced SIMS, 2,5-dihydroxybenzoic acid electrosprayed on neuroblastoma cells allowed intact molecular ion imaging of phosphatidylcholine and sphingomyelin at the cellular level. Gold deposition on top of matrix-coated rat brain tissue sections strongly enhanced image quality and signal intensity in stigmatic matrix-assisted laser desorption/ionization imaging mass spectrometry. High-quality total ion count images were acquired, and the neuropeptide vasopressin was localized in the rat brain tissue section at the hypothalamic area around the third ventricle. Although the mechanism of signal enhancement by gold deposition is under debate, the results we have obtained for cells and tissue sections illustrate the potential of this sample preparation technique for biomolecular surface imaging by mass spectrometry.

Animals↗

PubMed2006

Combined infrared multiphoton dissociation and electron-capture dissociation using co-linear and overlapping beams in Fourier transform ion cyclotron resonance mass spectrometry.

A novel set-up for Fourier transform ion cyclotron resonance mass spectrometry (FTICR) is reported for simultaneous infrared multiphoton dissociation (IRMPD) and electron-capture dissociation (ECD). An unmodified electron gun ensures complete, on-axis overlap between the electron and the photon beams. The instrumentation, design and implementation of this novel approach are described. In this configuration the IR beam is directed into the ICR cell using a pneumatically actuated mirror inserted into the ion-optical path. Concept validation was made using different combinations of IRMPD and ECD irradiation events on two standard peptides. The ability to perform efficient IRMPD, ECD and especially simultaneous IRMPD and ECD using lower irradiation times is demonstrated. The increase in primary sequence coverage, with the combined IRMPD and ECD set-up, also increases the confidence in peptide and protein assignments.

Amino Acid Sequence↗

PubMed2006

The molecular scanner in microscope mode.

The combination of microscope mode matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) with protein identification methodology: the molecular scanner, was explored. The molecular scanner approach provides improvement of sensitivity of detection and identification of high-mass proteins in microscope mode IMS. The methodology was tested on protein distributions obtained after separation by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). High-quality, high-spatial-resolution ion images were recorded on a TRIFT-II ion microscope after gold coating of the MALDI sample preparation on the poly(vinylidenedifluoride) capture membranes. The sensitivity of the combined method is estimated to be 5 pmol. The minimum amount of sample consumed, needed for identification, was estimated to be better than 100 fmol. Software tools were developed to analyze the spectral data and to generate broad mass range and single molecular component microscope mode ion images and single mass-to-charge ratio microprobe mode images.

Animals↗

PubMed2005

Monitoring macromolecular complexes involved in the chaperonin-assisted protein folding cycle by mass spectrometry.

We have used native mass spectrometry to analyze macromolecular complexes involved in the chaperonin-assisted refolding of gp23, the major capsid protein of bacteriophage T4. Adapting the instrumental methods allowed us to monitor all intermediate complexes involved in the chaperonin folding cycle. We found that GroEL can bind up to two unfolded gp23 substrate molecules. Notably, when GroEL is in complex with the cochaperonin gp31, it binds exclusively one gp23. We also demonstrated that the folding and assembly of gp23 into 336-kDa hexamers by GroEL-gp31 can be monitored directly by electrospray ionization mass spectrometry (ESI-MS). These data reinforce the great potential of ESI-MS as a technique to investigate structure-function relationships of protein assemblies in general and the chaperonin-protein folding machinery in particular. A major advantage of native mass spectrometry is that, given sufficient resolution, it allows the analysis at the picomole level of sensitivity of heterogeneous protein complexes with molecular masses up to several million daltons.

Bacteriophage T4↗

PubMed2005

Direct molecular imaging of Lymnaea stagnalis nervous tissue at subcellular spatial resolution by mass spectrometry.

The imaging capabilities of time-of-flight secondary ion mass spectrometry (ToF-SIMS) and MALDI-MS sample preparation methods were combined. We used this method, named matrix-enhanced (ME) SIMS, for direct molecular imaging of nervous tissue at micrometer spatial resolution. Cryosections of the cerebral ganglia of the freshwater snail Lymnaea stagnalis were placed on indium-tin-oxide (ITO)-coated conductive glass slides and covered with a thin layer of 2,5-dihydroxybenzoic acid by electrospray deposition. High-resolution molecular ion maps of cholesterol and the neuropeptide APGWamide were constructed. APGWamide was predominantly localized in the cluster of neurons that regulate male copulation behavior of Lymnaea. ME-SIMS imaging allows direct molecule-specific imaging from tissue sections without labeling and opens a complementary mass window (<2500 Da) to MALDI imaging mass spectrometry at an order of magnitude higher spatial resolution (<3 microm).

Animals↗

PubMed2005

Subcellular imaging mass spectrometry of brain tissue.

Imaging mass spectrometry provides both chemical information and the spatial distribution of each analyte detected. Here it is demonstrated how imaging mass spectrometry of tissue at subcellular resolution can be achieved by combining the high spatial resolution of secondary ion mass spectrometry (SIMS) with the sample preparation protocols of matrix-assisted laser desorption/ionization (MALDI). Despite mechanistic differences and sampling 10(5) times less material, matrix-enhanced (ME)-SIMS of tissue samples yields similar results to MALDI (up to m/z 2500), in agreement with previous studies on standard compounds. In this regard ME-SIMS represents an attractive alternative to polyatomic primary ions for increasing the molecular ion yield. ME-SIMS of whole organs and thin sections of the cerebral ganglia of Lymnaea stagnalis demonstrate the advantages of ME-SIMS for chemical imaging mass spectrometry. Subcellular distributions of cellular analytes are clearly obtained, and the matrix provides an in situ height map of the tissue, allowing the user to identify rapidly regions prone to topographical artifacts and to deconvolute topographical losses in mass resolution and signal-to-noise ratio.

Animals↗

PubMed2005

Proteome imaging: a closer look at life's organization.

Imaging the proteome is a term that is used in many different contexts. The term implies that the entire cohort of proteins and their modifications are visualized. This unfortunately is not the case. In this mini-review, a concise overview is provided on different imaging technologies that are currently used to investigate the structure, function and dynamics of proteins and their organization. These techniques have been selected for review based on the unique insights they provide in subsets of the proteome. These techniques have been illustrated with practical examples of their merits. Mass spectrometry-based imaging technologies are playing a key role in proteome research and have been reviewed in more detail. They hold the promise of detailed molecular insight in the spatial organization of living system.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2005

Does double electron capture lead to the formation of biradicals? An ECD-SORI-CID study on lacticin 481.

We studied lacticin 481, a small lantibiotic with three lanthionine bridges, by electron capture dissociation (ECD) in a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Following electron capture, very little fragmentation was observed, but species formed by nondissociative single and multiple electron capture were abundant. Ions formed by double electron capture were subjected to sustained off resonance irradiation collision induced dissociation (SORI-CID) to determine whether stable biradicals were formed. In the SORI-CID spectra of the ions formed by double electron capture, some, but minor, H* radical loss was observed, which was not observed at all for regularly protonated ions. A small part of the ions formed by double electron capture are thus long-lived biradicals. Apart from the observed H* loss, the SORI-CID spectra of ions that captured two electrons was similar to that of regularly protonated ions and quite different from the SORI-CID spectra of radical ions formed by single electron capture. This implies that recombination of the two radical sites is the dominant process in biradical lacticin 481 ions, at least on the time scale of our SORI-CID experiments.

Bacteriocins↗

PubMed2004

High-spatial resolution mass spectrometric imaging of peptide and protein distributions on a surface.

For the first time macromolecular ion microscope images have been recorded using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Single-shot, mass-resolved images of the spatial distributions of intact peptide and protein ions over an area of 200 microm in diameter were obtained in less than 1 ms at a repetition rate of 12 Hz. The magnifying ion optics of the ion microscope allowed ion images to be obtained with a lateral resolution of 4 microm. These results prove the concept of high-resolution MALDI-MS imaging in microscope mode without the need for a tight laser focus and the accompanying sensitivity losses. The ion microscopy approach offers an improvement of several orders of magnitude in speed of acquisition compared to the conventional (microprobe) approach to MALDI-MS imaging.

Image Processing, Computer-Assisted↗

PubMed2004

Electron capture dissociation at low temperatures reveals selective dissociations.

Electron capture dissociation at 86 K of the linear peptide Substance P produced just two backbone fragments, whereas at room temperature eight backbone fragments were formed. Similarly, with the cyclic peptide gramicidin S, just one backbone fragment was formed at 86 K but five at room temperature. The observation that some backbone scissions are active and others inactive, when all involve NC(alpha) cleavages and have a high rate constant, indicates that the more specific fragments at low temperatures reflects the reduced conformation heterogeneity at low temperatures. This is supported by reduced or inactive hydrogen loss, a channel that has previously been shown to be affected by conformation. The conclusion that the ECD fragments are a snapshot of the conformational (intramolecular solvation shell) heterogeneity helps explain how the relative intensities of ECD fragments can be different on different instrument and highlights the common theme in methodologies used to increase sequence coverage, namely an increase in the conformational heterogeneity of the precursor ion population.

Cold Temperature↗

PubMed2003

Using matrix peaks to map topography: increased mass resolution and enhanced sensitivity in chemical imaging.

It is well known in secondary ion mass spectrometry (SIMS) that sample topography leads to decreased mass resolution. Specifically, the ion's time of flight is dependent on where it was generated. Here, using matrix-enhanced SIMS, it is demonstrated that, in addition to increasing the yield of intact pseudomolecular ions, the matrix allows the user to semiquantitatively record the topography of a sample. Through mapping the topography-related mass shifts of the matrix (which leads to decreased mass resolution), the analogous mass shifts of higher mass ions can be deconvoluted and higher resolution and greater sensitivity obtained. Furthermore, the semiquantitative topographical map obtained can be compared with any chemical images obtained, allowing the user to quickly ascertain whether local intensity maximums are due to topological features or represent genuine features of interest.

Journal Article↗

PubMed2003

Localization of intramolecular monosulfide bridges in lantibiotics determined with electron capture induced dissociation.

Electron capture induced dissociation (ECD) and collisionally activated dissociation (CAD) experiments were performed on four lanthionine bridge-containing antibiotics. ECD of lantibiotics produced mainly c and z* ions, as has been observed previously with other peptides, but more interestingly, the less common c* and z ions were observed in abundance in the ECD spectra. These fragments specifically resulted from the cleavage of both a backbone amine bond and the thioether bond in a lanthionine bridge. ECD seemed to induce mainly cleavages near the lanthionine bridges. This fragmentation pattern indicates that lanthionine bridges play a key role in the selectivity of the ECD process. A new mechanism is postulated describing the formation of c* and z ions. Comparative low-energy CAD did not show such specificity. Nondissociative ECD products were quite abundant, suggesting that relatively stable double and triple radicals can be formed in the ECD process. Our results suggest that ECD can be used as a tool to identify the C-terminal attachment site of lanthionine bridges in newly discovered lantibiotics.

Amino Acid Sequence↗

PubMed2003

Effect of local matrix crystal variations in matrix-assisted ionization techniques for mass spectrometry.

Intense intact molecular ion signals have been obtained from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidyiinositol using matrix-enhanced secondary ion mass spectrometry (ME-SIMS). It was found that the high-mass (m/z >500) regions of the ME-SIMS spectra closely resembled those obtained using matrix-assisted laser desorption/ionization (MALDI). Using high spatial resolution SIMS, a detailed investigation of dried-droplet samples was performed. Based on the detected Na+ and 2,5-DHB matrix signal intensities, different crystal types were distinguished, in addition to different sizes of crystals. Spatially mapping the pseudomolecular and fragment ions of the phospholipids revealed that the nature of the pseudomolecular ions formed, as well as the ratio of intact molecular to fragment ion, was dependent on the type and surface composition of the crystal. The observed chemical bias effects due to crystal heterogeneity and the resulting variation in desorption/ionization efficiency will complicate the interpretation of data obtained from matrix-assisted mass spectrometric (imaging) techniques and is an important factor in the "hot spot" phenomenon frequently encountered in MALDI experiments. In this respect, imaging SIMS was found to be a versatile tool to investigate the effects of the local physicochemical conditions on the detected molecular species.

Crystallization↗