MALDI MS Tissue Imaging of Crystallins using an original metyhod to direct protein identification on lens slices

Poster (2010, April 16)

The lens is a transparent, biconvex structure in the eye that, along with the cornea, helps to refract light to be focused on the retina. Crystallins, α, β and γ, are the predominant structural proteins ... [more ▼]

The lens is a transparent, biconvex structure in the eye that, along with the cornea, helps to refract light to be focused on the retina. Crystallins, α, β and γ, are the predominant structural proteins in lens. They constitute 90% of water soluble proteins and contribute to its transparency and refractive properties by a uniform concentration gradient in the lens. Nevertheless, if these crystallins undergo post translational modifications, they become less soluble and the opacity of eye lens increases. This phenomenon defines cataract. Yet, the nature and the mechanism of occurring of these modifications and how they happen are not fully understood. MALDI mass spectrometry imaging is a recent technique allowing examining proteins in their native location without the need for traditional processing methods such as extraction, homogenization, and separation. Nevertheless, one main difficulty lies in the identification of the detected species, especially proteins. MALDI-In Source Decay (MALDI-ISD) is a fragmentation process occurring in the mass spectrometer ion source. When the analyzed sample is a protein, ISD fragmentation leads to b-, c- and z-ions series, which allows for some sequencing of the protein. One great advantage of ISD is its fastness and easiness to be implemented since there is no need for a special treatment of the sample. The only requirement is the use of “ISD-favourable MALDI matrix” such as 2,5-dihydroxybenzoic acid or 1,5-diaminonaphtalene. 18 µm-thick equatorial sections of frozen porcine eye lenses were realized with a cryostat. 1,5-DAN matrix was either manually deposited or sprayed with an ImagePrep automated device (Bruker Daltonics). Data were acquired with an UltraFlex II MALDI-TOF/TOF mass spectrometer (BD) in positive reflector mode. For imaging experiments, the surface of the sample was divided into 100-µm-wide pixels and 500 shots were averaged on each. Based on calculated mass differences between consecutive ISD fragments peaks, tags of amino acids were established and submitted to a search in protein databases using a BLAST algorithm (search by sequence homology). Imaging experiments showed that the localization information may be very useful to associate fragments which exhibit close distributions, suggesting they are originating from the same protein. It is thus possible to arrange fragments in groups of probable origin and to extract the mass spectrum of a high-intensity pixel. This allows to work with a “purified” ISD mass spectrum where fragments of only one protein are present and potentially exhibiting a higher number of peaks, leading to a longer tag and to an easier identification. With this imaging strategy, we were able to identify (by homology) the Beta-Crystallins S and B2, the Gamma-Crystallin B, the Alpha-Crystallin A. [less ▲]

GCXGC coupled to fast scanning quadrupole MS for trace analysis of POPs

in Book of abstracts (2010, January)

Dynamics of the Dictyostelium discoideum mitochondrial proteome during vegetative growth, starvation and early stages of development

in Proteomics (2010), 9

In this study a quantitative comparative proteomics approach has been used to analyze the D. discoideum mitochondrial proteome variations during vegetative growth, starvation and the early stages of ... [more ▼]

In this study a quantitative comparative proteomics approach has been used to analyze the D. discoideum mitochondrial proteome variations during vegetative growth, starvation and the early stages of development. Application of 2D-DIGE technology allowed the detection of around 2000 protein spots on each two-dimensional gel with 180 proteins exhibiting significant changes in their expression level. In total, 96 proteins (51 unique and 45 redundant) were unambiguously identified. We show that the D. discoideum mitochondrial proteome adaptations mainly affect energy metabolism enzymes (the Krebs cycle, anaplerotic pathways, the oxidative phosphorylation system and energy dissipation), proteins involved in developmental and signalling processes as well as in protein biosynthesis and fate. The most striking observations were the opposite regulation of expression of citrate synthase and aconitase and the very large variation in the expression of the alternative oxidase (AOX) that highlighted the importance of citrate and AOX in the physiology of the development of D. discoideum. Mitochondrial energy states measured in vivo with MitoTracker Orange CMTMRos showed an increase in mitochondrial membrane polarisation during D. discoideum starvation and starvation-induced development. [less ▲]

Development of quantitative methods to detect trace amounts of hazelnut and soy allergens in food

Poster (2010)

Behavioural effects of Endosulfan on amphibian tadpoles (Rana temporaria) using video-tracking and visual techniques

Poster (2010)

Internal energy content of ions in a travelling wave ion guide

Conference (2010)

Travelling wave ion guides (TWIGs) separate of ions according to their mobility and, at constant charge, according to their shape. The ion mobility separation itself should not modify the shape of the ... [more ▼]

Travelling wave ion guides (TWIGs) separate of ions according to their mobility and, at constant charge, according to their shape. The ion mobility separation itself should not modify the shape of the systems investigated. It was recently suggested by Shvartsburg et al. that the higher fields used in TWIGs than in traditional drift tubes would cause significant heating of the ions. We present a quantitative analysis of the amount of internal energy imparted to ions as they are separated in TWIGs. Benzylpyridinium ions were chosen as “thermometer” ions. Based on arrival time distributions, the fragment ion population is separated as a function of the place of formation: before, in, or after the TWIG. Fragmentation all along the TWIG was observed. The roles of the travelling wave’s voltage, speed, and of the gas nature and pressure will be discussed in detail as well of the consequences on instruments performances. [less ▲]

Furan formation in baby food model systems from vitamin C and unsaturated fatty acids

Conference (2010)

Cation Involvement in Telomestatin Binding to G-Quadruplex DNA

in Journal of Nucleic Acids (2010)

The binding mode of telomestatin to G-quadruplex DNA has been investigated using electrospray mass spectrometry, by detecting the intact complexes formed in ammonium acetate. The mass measurements show ... [more ▼]

The binding mode of telomestatin to G-quadruplex DNA has been investigated using electrospray mass spectrometry, by detecting the intact complexes formed in ammonium acetate. The mass measurements show the incorporation of one extra ammonium ion in the telomestatin complexes. Experiments on telomestatin alone also show that the telomestatin alone is able to coordinate cations in a similar way as a crown ether. Finally, density functional theory calculations suggest that in the G-quadruplex-telomestatin complex, potassium or ammonium cations are located between the telomestatin and a G-quartet. This study underlines that monovalent cation coordination capabilities should be integrated in the rational design of G-quadruplex binding ligands. [less ▲]

Isotope coded protein label quantification of serum proteins--comparison with the label-free LC-MS and validation using the MRM approach.

in Talanta (2010), 80(4), 1487-95

Protein quantification based upon mass spectrometry is gaining ground in diverse applications of biological and clinical relevance. The present article focuses on one of the most complex biological fluids ... [more ▼]

Protein quantification based upon mass spectrometry is gaining ground in diverse applications of biological and clinical relevance. The present article focuses on one of the most complex biological fluids - serum - and provides a novel ICPL based quantification protocol. The results are compared to a label-free (data independent alternate scanning) absolute quantification method. The validation is performed using MRM based protein quantification technique. Regarding the ICPL approach, serum samples used in this study were depleted of high abundant proteins, labeled with ICPL and fractionated according to their respective pI (3-5, 5-7 and 7-12). The samples were further subjected to tryptic digestion followed by treatment with the Glu-C enzyme. The peptides were analyzed on a 2D-nano-LC system using four different concentrations of salt injections (45, 75, 150 and 500 mM ammonium acetate). The LC system was connected on-line with the electrospray ion-trap mass spectrometer. For the label-free quantification the serum samples were depleted and digested with trypsin. A proteome-wide comparison was performed using highly reproducible LC and data independent alternate scanning in conjunction with a high mass accuracy orthogonal time-of-flight mass spectrometer. Selected proteins, found by both methods, were validated using the MRM approach. For this purpose non-depleted tryptically digested serum samples were analyzed by LC coupled with a triple-quadrupole MS. The relative protein quantification using ICPL and mass spectrometry allowed for the detection of approximately 200 proteins, whereas about 2/3 of those contained the ICPL label and could therefore be quantified. Label-free approach used no fractionation, less sample and was able to identify and quantify over 110 proteins. The identified proteins covered generally 3-4 orders of magnitude of protein concentration in human serum. Changes in relative abundance of eight proteins were validated using MRM. This study, for the first time, shows the ability of the relative protein quantification based upon ICPL and 2D-LC-MS/MS to quantify serum biomarkers. It provides two additional label-free approaches that could validate and bring additional value to the label-based results, offering a starting point for comprehensive proteomics studies aiming at revealing biomarkers of clinical relevance. [less ▲]

Identification of Fragmentation Channels of Dinucleotides Using Deuterium Labeling.

in Journal of the American Society for Mass Spectrometry (2010), 21(1), 23-33

The fragmentation of the totally deuterated dinucleotide dAT(-) in labile positions (heteroatom-bound hydrogens) was compared for different MS/MS methods: CID, IRMPD, and EID. These experiments allowed us ... [more ▼]

The fragmentation of the totally deuterated dinucleotide dAT(-) in labile positions (heteroatom-bound hydrogens) was compared for different MS/MS methods: CID, IRMPD, and EID. These experiments allowed us to affirm the coexistence of several fragmentation channels. They can be classified according to the involvement of nonlabile or labile protons in the fragmentation process. Moreover, double resonance experiments were performed in IRMPD and EID. They demonstrated the existence of consecutive fragmentation processes. The probability with which each channel is taken depends on the fragmentation technique used, i.e., the energy and the time scale of the method. The fragmentation channels that involve labile protons requiring peculiar three-dimensional structures are entropically unfavorable and enthalpically favorable. They are more observed in IRMPD and EID. The involvement of labile and, therefore, exchangeable protons in the fragmentation mechanism casts doubt on the use of tandem mass spectrometry to localize incorporated deuteriums in oligonucleotides. [less ▲]