Mass spectrometry fundamentals

New fragmentation methods involving radical ions

Traditional fragmentation techniques in MS/MS are based on collision-induced dissociation. In collisional activation of even-electron species, the internal energy spreads over all degrees of freedom before fragmentation. However, odd-electron species leads to very reactive intermediates and very particular fragmentation patterns. These methods include electron capture dissociation (ECD) electron-detachment dissociation (EDD), in-source decay (ISD) in MALDI, and UV photofragmentation.

Mass spectrometry of nucleic acids structures and complexes

Electrospray mass spectrometry has now proven to be a valid method for the study of DNA and DNA complexes with small molecules (determination of the stoichiometry and of binding constants). The DNA structures include Watson-Crick duplexes, but also triplexes and quadruplexes, which are relevant targets in oncology. We are trying to investigate whether these DNA structures and their complexes with small ligands are conserved in the gas phase, using ion mobility spectrometry, H/D exchange and special fragmentation techniques.

Biochemistry and biophysics

Ligand disruption of protein aggregation processes in Alzheimer’s disease

The origin of many neurodegenerative diseases like Alzheimer’s disease lies in protein processing failures. The proteins aggregate and accumulate as amyloid fibrils. Understanding the aggregation mechanism and how to prevent aggregate formation is therefore crucial. Here we use electrospray ionization mass spectrometry to measure ligand binding to proteins, and the inhibition of protein aggregation that follows ligand binding. We also apply various MS techniques to characterize ligand binding mode to the proteins.

Electrospray-MS screening of DNA-targeting molecules

Electrospray ionization mass spectrometry allows the detection of intact noncovalent complexes, determine their stoichiometry, and the binding constants. Kinetic studies in the second to hour time scale can also be performed, by monitoring the relative intensities as a function of time. Our major application of these studies is in the field of drug-DNA interactions. The DNA target we study are DNA duplexes, triplexes, quadruplexes, mismatched duplexes,… Molecular modeling is used for the rationalization of the structural preference of some classes of drugs.

Protein response to cell treatment by DNA-targeting ligands

We compare the activity of therapeutic agents targeting specifically G-quadruplex DNA at the molecular and at the cellular level. We are looking for correlations between the binding properties of the drugs (affinity constant, structural specificity), the protein expression levels, and perturbations of the cell cycles. The current projects includes the developement of sample preparation methods that capture specifically quadruplex-binding proteins, the comparison of hTERT+ and hTERT- cell lines, and the comparisons between treated and untreated cell lines.

Photo-oxidation of DNA polyanions by UV-vis laser irradiation

We recently have demonstrated that irradiation of DNA anions by UV light (260-280 nm) provokes the ejection of electrons, i.e. DNA oxidation. We are currently studying the base-dependence, sequence-dependence and conformation-dependence of this process. Photo-oxidation leads to long-lived DNA radicals containing holes, and we can investigate hole migration and hole trapping with MS/MS fragmentation techniques. These studies have important implications in DNA excited state dynamics, and in DNA photostability.

Analytical chemistry

Validated Predictive Dynamic Model of Complex Intracellular Pathways Related to Cell Death and Survival (VALAPODYN)

The search for early medication to reduce neurodegeneration is a major challenge in Neuroscience. We use a mouse model of induced hippocampal cell loss associated with focal epilepsy (INSERM). Ddynamic protein expression analyses are established at successive time-points post-induction. Extracted proteins are analysed by gel-based method (2 Dimensional – Differential In Gel Electrophoresis) and are identified by MALDI-TOF/TOF technique. These time-dependant networks will help to design functional modeling of the molecular aspects of neurodegeneration.

LC-MALDI-MS/MS strategies for quantitative analysis

MALDI techniques are the fastest way to identify proteins purified from genome-sequenced species. With automated possibilities, they allow the identification of a protein in less than a minute (provided that the sample is clean and already spotted on the MALDI target!). Intact proteins are analysed with in-source decay. We also investigate how to apply MALDI-MS/MS techniques to the identification and quantification of small molecules. This includes the evaluation of new matrices in terms of ionization and fragmentation efficiency.

Proteomics tools to sceen multiple contaminants in food

Identification of candidate biomarkers using a range of proteomic techniques and initiation of biosensor assay development work. The selected biomarkers will be used in diagnostic assay to detect for the presence of fingerprints in sample taken from cattle treated with a wide range of anabolic steroids. The assay should be capable of detecting the presence of biomarkers for at least a period of two weeks following the end of the medication period. The assay should also not identify more than 10% of untreated cattle as ‘biomarker positive’.

Analysis of dioxins and PCBs in food and feed

The laboratory is accredited for dioxin and PCB analysis in several matrices like food, milk, blood,… We participated to food analysis campaigns since the belgian dioxin crisis in 1999, and contributed to the development efficient sample preparation techniques for faster analyses. We use high resolution sector instruments and ion trap mass spectrometers. Recently, the lab performed a toxicokinetic study of dioxin in laying chicken, and also participated to a study of human exposure to dioxins and PCBs in the neighborhood of an incinerator.

Analysis of doping agents using proteomics techniques

There is actually no approved method for the detection of human Growth Hormone belonging to the doping agent’s classes of peptide hormones, and a fortiori no approved method for simultaneous detection of this whole class. The aim of this project is to develop new methods using proteomics techniques. The first step is the search for suitable biomarkers of these doping agents: in some cases a biomarker will be preferred to the detection of the doping agent itself. We use isotope dilution by heavy peptides and MRM strategies for the quantification.

Aptamer-based strategies for selective sample preparations

Aptamers are artificial oligonucleotides ligands that have been selected against amino acids, proteins or small molecules. They are isolated from complex libraries of synthetic nucleic acid using Selective Expansion of Ligands by Exponential Enrichment (SELEX) and are very specific towards their target. We are working with aptamers targeted towards antibiotics to develop analytical methods for the sample preparation. This would allow to isolate the desired antibiotic from complex matrices and improve the detection limits.

Development of biosensors

With the development of proteomics and genomics comes the need for tools allowing for rapid detection of characteristic biomolecules. One research area of great interest is the development of molecular nano-scale sensors. Our goal is to develop a new sensor for fluorescence and NMR detection of biomolecules. Mass spectrometry is used in the present project to characterize and study some physico-chemical properties of the assembly constituting the sensor. Our work includes modeling, synthesis, characterization and validation.

Investigation of plant – aphid – bacterial endosymbionts interactions

Proteomics is used to determine the proteome patterns related to differential adaptations and metabolic changes of aphids to cope with plant defense mechanisms, with a focus on the role of the bacterial endosymbionts. The proteome comparison of insects reared on different host plants and the use of artificial diets including diverse molecules are studied to identify the respective origin (symbionts or insect itself) and function of the identified proteins. Proteomics is an interesting and reliable tool to study insect – host plant interactions.