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Biomedical subjects

Renato Zenobi

Publications and source records attributed to Renato Zenobi.

At least 19 recordsLinked to original sources
PubMed2007

Proteomic profiling of a snake venom using high mass detection MALDI-TOF mass spectrometry.

Proteomic profiling involves identification and quantification of protein components in complex biological systems. Most of the mass profiling studies performed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been restricted to peptides and small proteins (<20 kDa) because the sensitivity of the standard ion detectors decreases with increasing ion mass. Here we perform a protein profiling study of the snake venom Sistrurus miliarius barbouri, comparing 2D gel electrophoresis and reversed-phase high-performance liquid chromatography (HPLC) with a high mass cryodetector MALDI-TOF instrument (Macromizer), whose detector displays an uniform sensitivity with mass. Our results show that such MS approach can render superior analysis of protein complexity compared with that obtained with the electrophoretic and chromatographic approaches. The summation of ion impacts allows relative quantification of different proteins, and the number of ion counts correlates with the peak areas in the reversed-phase HPLC. Furthermore, the sensitivity reached with the high mass cryodetection MS technology clearly exceeds the detection limit of standard high-sensitivity staining methods.

Amino Acid Sequence↗
PubMed2006

Shrinking droplets in electrospray ionization and their influence on chemical equilibria.

We investigated how chemical equilibria are affected by the electrospray process, using simultaneous in situ measurements by laser-induced fluorescence (LIF) and phase Doppler anemometry (PDA). The motivation for this study was the increasing number of publications in which electrospray ionization mass spectrometry is used for binding constant determination. The PDA was used to monitor droplet size and velocity, whereas LIF was used to monitor fluorescent analytes within the electrospray droplets. Using acetonitrile as solvent, we found an average initial droplet diameter of 10 microm in the electrospray. The PDA allowed us to follow the evolution of these droplets down to a size of 1 microm. Rhodamine B-sulfonylchloride was used as a fluorescent analyte within the electrospray. By spatially resolved LIF it was possible to probe the dimerization equilibrium of this dye. Measurements at different spray positions showed no influence of the decreasing droplet size on the monomer-dimer equilibrium. However, with the fluorescent dye pair DCM and oxazine 1 it was shown that a concentration increase does occur within electrosprayed droplets, using fluorescence resonance energy transfer as a probe for the average pair distance.

Journal Article↗
PubMed2006

Immunoassays with direct mass spectrometric detection.

A rapid, specific, and sensitive method for the detection of protein-protein interactions is of crucial importance for drug discovery and clinical diagnostics. Mass spectrometry plays a major role in the analysis of proteins, but its application to the routine analysis of protein complexes has been lagging behind. A new strategy for high-throughput analysis of protein interactions is presented here. We demonstrate application to immunochemical questions such as epitope mapping, kinetic studies, and sandwich assays. The methodology is based on a direct mass spectrometric readout for antigen-antibody complexes in the 150-400 kDa range. This has become possible using a novel detector technology and chemical cross-linking to stabilize complexes for analysis by MALDI MS. We demonstrate high detection sensitivity (femtomole quantities of antigen), high specificity (specific detection of antigen directly in serum), high accuracy, and high speed (minutes per assay), surpassing conventional analytical methods by more than 2 orders of magnitude.

Animals↗
PubMed2006

Effect of buffer gas on the fluorescence yield of trapped gas-phase ions.

We investigated the dependence of three different gases, helium, argon, and nitrogen, on the fluorescence signal intensity of rhodamine 6G cations in the gas phase. The method is based on laser-induced fluorescence of ions trapped in a Fourier transform ion cyclotron mass spectrometer. We found that the use of helium results in the highest fluorescence signal, while no fluorescence was detected when using argon under the same conditions.

Journal Article↗
PubMed2006

Kinetic energy of free electrons affects MALDI positive ion yield via capture cross-section.

A method for enhancing positive analyte ion signal in MALDI is described. The idea is based on influencing the kinetic energy of free electrons emitted from the organic/metal interface. It has been recently shown that free electrons in MALDI have energies around 1 eV. This energy is close to the maximum capture cross-section of most common MALDI matrices, leading to the efficient formation of negative matrix ions. This results in the reduction of the positive analyte ion yield. The effect can be minimized by shifting the kinetic energy of the electrons away from the maximum of the matrix capture cross-section by choosing a different substrate material.

Electrons↗
PubMed2006

Detection of noncovalent complexes in biological samples by intensity fading and high-mass detection MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has not yet contributed widely to the study of intact noncovalent biomolecular complexes, because MALDI is known to cause dissociation of the interaction partners and induce formation of nonspecific aggregates. Here, we present a new strategy to circumvent this problem. It is based on intensity fading (in the low m/z range) and high-mass detection MALDI mass spectrometry (MS), using a cryodetector (in the high m/z range), with and without chemical cross-linking of the interaction partners. The study focuses on noncovalent interactions between the human enzyme carboxypeptidase A (hCPA) and three protease inhibitors (PCI, TCI, and LCI) present in heterogeneous mixtures of other nonbinding molecules derived from a biological source, an extract from leech (Hirudo medicinalis). Another example involves an extract of the sea anemone Stichodactyla helianthus, which is used without previous fractionation to detect the specific complex between the enzyme trypsin and the endogenous SphI-1 inhibitor. The results give insight into the mechanism of intensity fading MS and demonstrate that the specificity of binding is greatly favored when the overall concentrations of the analytes (nonbinding molecules, protease inhibitor and target enzyme) present in a biological sample of interest are kept at low concentrations, in the sub-micromolar range. Higher concentrations may lead to unspecific interactions and the formation of aggregates both during the MALDI process and during reaction with the cross-linking reagents. This strategy is expected to advance the field of high-throughput affinity-based approaches, by taking advantage of a new generation of high mass detectors for MALDI-TOF instruments.

Animals↗
PubMed2006

Enhancement of Raman signals with silver-coated tips.

Silver coated SiN and SiO(2) tips have been fabricated for use with a bottom-illumination tip-enhanced Raman spectroscopy (TERS) setup with a 488 nm laser excitation. SiN tips with 50-60 nm of deposited Ag give the best TERS enhancements for brilliant cresyl blue test analyte spin-coated on a glass slide. Ag nanoparticles on SiN or SiO(2) rather than Si tips are better for TERS because of the proximity of the wavelengths of their surface plasmon resonance to 488 nm. Adjustments of tilt angle of the metallized tip with respect to the surface plane is shown to considerably raise the intensities of the TERS signals, even from tips that initially appear to be rather non-enhancing. This work helps to enable the more frequent use of the 488 nm laser for nanoscale chemical analysis with both TERS and fluorescence imaging in the same setup.

Microscopy, Scanning Probe↗
PubMed2005

Interfacing liquid chromatography with atmospheric pressure MALDI-MS.

Two different strategies for coupling liquid chromatography with atmospheric pressure matrix assisted laser desorption/ionization (AP MALDI) are presented. The first method is flow-injection liquid AP UV-MALDI. Compared with previous similar research, the detection limit was improved 10 times to 8.3 fmol using a solution of 50 nM peptide with 25 mM alpha-cyano-4-hydroxycinnamic acid. The applicability of this method to measure oligosaccharides, actinomycin antibiotics, antibiotics, phosphopeptides, and proteins is demonstrated. The upper mass limit achieved with the current instrumentation is 6,500 Da (doubly charged cytochrome c). The feasibility of a second strategy based on single-droplet IR AP MALDI is demonstrated here. Aqueous peptide solutions were successfully measured by this method.

Atmospheric Pressure↗
PubMed2005

Analysis of megadalton ions using cryodetection MALDI time-of-flight mass spectrometry.

Presented are initial results from the first commercially available matrix-assisted laser desorption/ionization time-of-flight mass spectrometer specifically designed for the sensitive detection of very high mass ions (macromizer, Comet AG). This new instrument utilizes a 16-element superconducting tunnel junction detector coupled with a fully adjustable gimbal-mounted ion source/focusing region that allows unparalleled sensitivity for detection of singly charged high molecular weight ions. Using this new technology, the singly charged ions in the megadalton region are detected from immunoglobulin M and von Willebrand factor proteins. This detector technology also measures the kinetic energy of the particles impacting the detector, which can be correlated to the charge of the particles. Immunoglobulin G and streptavidin were used to demonstrate the ability of the macromizer instrument to detect high-mass ions and to discern the charge state of the ions.

Journal Article↗
PubMed2005

High time resolution and size-segregated analysis of aerosol bound polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are an ubiquitous class of compounds in the environment, mostly generated by anthropogenic processes. High time resolution measurements are necessary to gain further knowledge on the fate and diurnal pattern of these often carcinogenic and mutagenic compounds in the atmosphere. It is expected to find a strong correlation of the PAH levels with the strength and proximity to sources, as well as with meteorological parameters. To determine the fate of particle-bound PAHs, they were sampled in this study at an urban background site in Zurich, Switzerland, during summer 2002 and winter 2003. Particle-bound PAHs were collected with a rotating drum impactor and subsequently analyzed with two-step laser mass spectrometry. Using this combination of sampling and measurement, size-segregated (10-1.1, 1.1-0.3, and 0.3-0.1 microm) and high time resolution (20 min) data were obtained. The pronounced diurnal cycle (with day/night ratios of 0.1) was only altered during intensive atmospheric mixing periods (resulting in day/night ratios of up to 8) by cleaner air from upper atmospheric layers which was mixed into the boundary layer. During summer, signal intensities due to particle-bound PAHs were about a factor of 2-10 lower than during winter.

Aerosols↗
PubMed2005

Determination of zinc to beta-peptide binding constants with electrospray ionization mass spectrometry.

We present an improvement of the titration method for binding constant determination with electrospray ionization (ESI) mass spectrometry that is unaffected by differences in ESI response of measured species in solution. The method consists of a calibration and titration, both using an internal standard that allows relative quantitation. This avoids artifacts such as a decrease in overall signal intensity with increasing ligand concentrations, rendering this approach more reliable and meaningful than direct evaluation of ESI peak intensities. We demonstrate the de novo binding constant determination of novel zinc binding beta-peptides, which have been synthesized with the goal of creating secondary structures stabilized by metal complexation.

Oligopeptides↗
PubMed2005

Clear evidence of fluorescence resonance energy transfer in gas-phase ions.

Fluorescence resonance energy transfer (FRET) is a distance-sensitive method that correlates changes in fluorescence intensity with conformational changes, for example, of biomolecules in the cellular environment. Applied to the gas phase in combination with Fourier transform ion cyclotron resonance mass spectrometry, it opens up possibilities to define structural/conformational properties of molecular ions, in the absence of solvent, and without the need for purification of the sample. For successfully observing FRET in the gas phase it is important to find suitable fluorophores. In this study several fluorescent dyes were examined, and the correlation between solution-phase and gas-phase fluorescence data were studied. For the first time, FRET in the gas phase is demonstrated unambiguously.

Fluorescence Resonance Energy Transfer↗
PubMed2005

Laser-induced fluorescence of trapped gas-phase molecular ions generated by internal source matrix-assisted laser desorption/ionization in a Fourier transform ion cyclotron resonance mass spectrometer.

The combination of laser-induced fluorescence with mass spectrometry opens up new possibilities both for detection purposes and for structural studies of trapped biomolecular ions in the gas phase. However, this approach is experimentally very challenging, and only a handful of studies have been reported so far. In this contribution, a novel scheme for laser-induced fluorescence measurements of ions trapped inside a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer will be introduced. It is based on an open FT-ICR cell design, continuous wave axial excitation of the fluorescence, orthogonal photon collection by fiber optics, and single photon counting detection. Rhodamine 6G ions generated by an internal matrix-assisted laser desorption/ionization source were used to develop and test the set-up. Due to photobleaching processes, the excitation laser power and the observation time window have to be carefully optimized. An ion tomography method was used to align the excitation laser. Potential applications for studying the gas-phase structure of fluorescent biomolecular ions and for investigating fluorescence resonance energy transfer of donor-acceptor pairs will be presented.

Journal Article↗
PubMed2004

On-line atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry.

A new technique is presented for the coupling of atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) mass spectrometry with liquid delivery systems. Mass measurements of polymers and peptides are demonstrated using a co-dissolved matrix, e.g. alpha-cyano-4-hydroxycinnamic acid (HCCA). Improvements in terms of sensitivity are achieved by optimizing the shape und control of the exit capillary and by using a laser (355 nm) at a 1 kHz repetition rate. Two calibration experiments promise a good applicability of the presented coupling method for quantitative measurements. The limit of detection achieved so far is 500 nM for peptides in methanol solution containing 25 mM HCCA.

Animals↗
PubMed2004

Analysis of size-segregated aerosol-bound polycyclic aromatic hydrocarbons with high time resolution using two-step laser mass spectrometry.

Polycyclic aromatic hydrocarbons are known for their mutagenic and carcinogenic properties. They are mainly emitted into the atmosphere by anthropogenic, incomplete combustion sources. Their trends over the course of a day are of interest in air quality management. A new combination of methods has been developed for the qualitative monitoring of polycyclic aromatic hydrocarbons in ambient aerosols with high time and size resolution. This has been accomplished by combining sampling with a rotating drum impactor and the analysis two-step laser mass spectrometry (L2MS). A validation for this method was carried out. Essential features of these combined techniques are (i) continuous, automatic sampling and (ii) selective as well as sensitive analysis due to the low detection limits which can be achieved with the L2MS analysis instrument. Analysis of a field sample taken in downtown Zurich, Switzerland, underlines the usefulness of this combined method, for example for following diurnal cycles of polycyclic aromatic hydrocarbons.

Air Pollutants↗