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See detailProtein Identification and Quantification in Mixtures of highly-modified Proteins
Dobson, Rowan ULg

Doctoral thesis (2013)

The identification and quantification of proteins in highly-modified mixtures using proteomics has been performed. Two research projects have been undertaken which fulfil this aim. The first comprised the ... [more ▼]

The identification and quantification of proteins in highly-modified mixtures using proteomics has been performed. Two research projects have been undertaken which fulfil this aim. The first comprised the development of quantitative methods to detect trace amounts of hazelnut and soy in complex mixtures. A method for the detection and absolute quantification of Cor a 9, a major hazelnut (Corylus avellana) allergen was developed based on mass spectrometry. One hundred and ten hazelnut proteins were detected, five of which were allergens. The peptide chosen for quantification was from Cor a 9 (11S globulin-like protein). Two specific fragmentation reactions were chosen in multiplexed Selected Reaction Monitoring (SRM). Forty three hazelnut food processing imitation samples, varying a range of factors, such as the temperature and incubation time were analysed. A calibration curve was made for cookies. The developed method was for home-made cookies, shop-bought cookies and chocolate. The quantities of Cor a 9 in each sample were determined from the quantification of the target peptide using isotopic dilution with a heavy isotopically labelled peptide. A second peptide with two transitions was also proven to be a possible alternative as a detection method for hazelnut. The presence of soybean allergens in processed food can be detected using the mass spectrometric identification of a soybean peptide which is resistant to the heating and chemical reactions associated with food processing. The proteomic analysis of soybeans allowed the identification of 11 allergens. A method using a peptide (VFDGELQEGR) from glycinin G1 (Gly m 6.0101) was developed for the detection and semi-quantification of the allergen in food samples. Two specific fragmentation pathways were chosen in selected reaction monitoring for unambiguous identification of glycinin G1 and were: 575.3 903.4 Da and 575.3 788.4 Da. Sixteen imitation samples of processed food spiked with soybean were analyzed, where factors such as temperature and incubation time were varied, and the chosen transitions were detected. The developed method was specific for home-made cookies and a shop-bought biscuit. Semi-quantification from both cooked and uncooked cookies was demonstrated. The second comprised the identification and quantification of conotoxins in the venom of Conus textile by the use of isotope coded affinity tagging (ICAT) and label-free quantification. The extreme variety and complexity of the conotoxins has been insufficiently documented and this research demonstrates the varied nature of conotoxins found in different parts of the venom duct and their patterns of expression. Fifteen conotoxins, several with different post-translational modifications (PTMs), were identified and quantified. Distinctive patterns emerged, with the largest group of conotoxins increasing, then peaking in the central6 proximal part, before decreasing; whilst the second largest group peaked in the distal region, generally displaying nothing in the first parts. A new conotoxin, PCCSKLHDNSCCGL*, was sequenced. [less ▲]

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See detailSecretion and maturation of conotoxins in the venom ducts of Conus textile
Dobson, Rowan ULg; Collodoro, Mike; Gilles, Nicolas et al

in Toxicon (2012), 60(8), 1370-1379

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See detailIdentification and quantification of concentration-dependent biomarkers in MCF-7/BOS cells exposed to 17β-estradiol by 2-D DIGE and label-free proteomics
Collodoro, Mike ULg; Lemaire, Pascale ULg; Eppe, Gauthier ULg et al

in Journal of Proteomics (2012), in press

This paper reports the identification of biomarkers resulting from the exposure of MCF-7/BOS cells to 17β-estradiol (E2). The biomarkers were identified using 2 independent and complementary techniques, 2 ... [more ▼]

This paper reports the identification of biomarkers resulting from the exposure of MCF-7/BOS cells to 17β-estradiol (E2). The biomarkers were identified using 2 independent and complementary techniques, 2-D DIGE / MALDI-TOF peptide mass fingerprint, and 2-D UPLC-ESI MS/MS. These markers form a preliminary molecular signature that can be used when testing the estrogenic activity of xenobiotics, either pure or in mixtures. [less ▲]

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See detail2D DIGE, label free quantification, principal component and mass spectrometry analysis for biomarkers discovery in MCF-7/BOS cells exposed to 17β-estradiol and endocrine disruptors.
Collodoro, Mike ULg; Lemaire, Pascale; Eppe, Gauthier ULg et al

in Organohalogen Compounds (2011)

Endocrine system disruption has become a subject of great interest over the last few decades, since it has become evident that natural and also synthetic substances can mimic or reduce the activity of ... [more ▼]

Endocrine system disruption has become a subject of great interest over the last few decades, since it has become evident that natural and also synthetic substances can mimic or reduce the activity of endogenous hormones. Compounds with estrogenic activity are an important family of potential endocrine disruptors that have to be monitored either in the food chain or in the environment. Estrogens are known to induce or promote hormonal dependent cancers, to reduce sperm counts and fertility in men and generate the feminization of exposed wildlife populations. The rapid screening of unwanted chemicals in the food chain is beset by difficulties. The number of toxic compounds is very large and no universal method can cope with their diversity. In this work, emergent differential proteomic techniques are used to discover a set of biomarkers for the development of a multiple estrogen contaminants screening test. [less ▲]

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See detailDynamics of the Dictyostelium discoideum mitochondrial proteome during vegetative growth, starvation and early stages of development
Czarna, Malgorzata; Mathy, Grégory ULg; Mac Cord, Allan ULg et al

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 ▲]

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See detailSecretion and maturation of toxins in the venom duct of Conustextile
Dobson, Rowan ULg; Corbesier, Corine; Collodoro, Mike et al

Conference (2009, December 02)

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See detailRelative serum protein quantification based upon ICPL and 2D-LC-MS identifies potential frailty biomarkers in elderly patients
Turtoi, Andrei ULg; Mazzucchelli, Gabriel ULg; Dobson, Rowan ULg et al

Poster (2009, June 01)

This study shows the ability of the ICPL and nano-HPLC-MS/MS to perform relative quantification and identification of serum protein biomarkers. Frailty is a geriatric syndrome that is commonly associated ... [more ▼]

This study shows the ability of the ICPL and nano-HPLC-MS/MS to perform relative quantification and identification of serum protein biomarkers. Frailty is a geriatric syndrome that is commonly associated with the decline in multisystemic reserve, cognition and sensory capabilities. It is negatively influencing the outcome of a disease prolonging the patient’s recovery. The discrepancy between the actual and the biological age brings the uncertainty of predicting frailty in a given individual. This study is addressing the problem of finding suitable biomarkers that bear the ability to objectively predict frailty in elderly patients. It furthermore provides a robust method for reliable relative quantification of serumproteins. Serum samples used in this study were divided into six groups regarding the patient’s disease (hip-fracture, infection and cardiac decompensation) and frailty status (frail or robust). The individual sera were pooled and a volume of 20 µL was depleted of high abundantproteins. After labeling with ICPL (isotope coded protein label), serum proteins were fractionated according to their respective pI (0-3, 4-7 and 8-12). The samples were further subjected to tryptic digestion followed by the treatment with the Glu-C enzyme. The peptides were analyzed on the 2D-nano-HPLC system (Ulimate 3000®) using four different concentrations of salt injections (45, 75, 150 and 500 mM ammonium acetate). The HPLC system was connected on-line with the electrospray ion-trap mass spectrometer Esquire HCT ultra®. The relative protein quantification using ICPL and mass spectrometry allowed for comparison of six patient groups with respect to a standard sample. The latter represented a group of healthy old subjects. This technique allowed for the detection of approx. 200 proteins, whereas about 50 % of those contained the ICPL label and could therefore be quantified. The identified proteins covered 3 – 4 orders of magnitude ofprotein concentration in human serum. Several proteins displayed a significant modulation allowing for some preliminary conclusions to be drawn. At this point it can be stated that significantly elevated levels of C-reactive protein (factor 12) and alpha-1-antichimotrypsin (f. 4) proved to be potentially good indicators of frailty. Increased concentrations of alpha-1-microglobulin (f. 4) and alpha-2HS-glycoprotein (f. 2) have been found in the robust patients, whereas no significant concentration alteration could be detected in the frail groups. These results refer to the acute phase response since the samples were collected immediately after patient hospitalization. Current investigations addressing the later sampling times should shed more light on the suitability of these markers to predict frailty in elderly patients. At this stage it is obvious that although several markers are found to be in common for all the frail or robust patients, the disease status additionally complicates the biomarker signature. Therefore a more individualized approach should also be considered, where depending on the age and clinical findings a more defined group of markers should be selected to address the problem of frailty. [less ▲]

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See detailMaturation of toxins in the venom duct of conustextile
Dobson, Rowan ULg; Collodoro, Mike; Gilles, Nicolas et al

Poster (2009, June)

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See detailIdentification of biomarkers to estrogen exposure using MCF-7/BOS cell line exposed to 17β-estradiol and phytoestrogens
Collodoro, Mike ULg; Bertrand, Virginie ULg; Lemaire, Pascale ULg et al

Poster (2009, June)

Use of an estrogen responsive cell line and proteomic for biomarker discovery and the screening of xenoestrogen

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See detailDevelopment of a quantitative method to detect trace amounts of hazelnut and soy allergens in food
Dobson, Rowan ULg; Fourdrilis, Séverine; Kirsch, Stéphanie ULg et al

Conference (2009)

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See detailQuantitative methods for food allergens: a review.
Kirsch, Stéphanie ULg; Fourdrilis, Séverine ULg; Dobson, Rowan ULg et al

in Analytical and Bioanalytical Chemistry (2009), 395

The quantitative detection of allergens in the food chain is a strategic health objective as the prevalence of allergy continues to rise. Food allergenicity is caused by proteins either in their native ... [more ▼]

The quantitative detection of allergens in the food chain is a strategic health objective as the prevalence of allergy continues to rise. Food allergenicity is caused by proteins either in their native form or in forms resulting from food processing. Progress in mass spectrometry greatly opened up the field of proteomics. These advances are now available for the detection and the quantification of traces of allergenic proteins in complex mixtures, and complete the set of biological tests used until now, such as ELISA or PCR. We review methods classified according to their ability to simultaneously quantify and identify allergenic proteins and underline major advances in the mass-spectrometric methods. [less ▲]

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See detailS13.45 Chlamydomonas reinhardtii mitoproteome adaptation in response to inactivation of the energy-dissipating alternative oxidase 1 by RNA interference
Cloes, Marie ULg; Mathy, Grégory ULg; Cardol, Pierre ULg et al

in Biochimica et Biophysica Acta (BBA) - Bioenergetics, Volume 1777, Supplement 1, 19 July 2008, Page S99 (2008, July 18), 1777(Supplement 1), 99

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See detailApoptosis and cytolysis induced by giganteosides and hederacolchisides in HL-60 cells
Gerkens, Pascal; Dobson, Rowan ULg; tabatadze, Nino et al

in Anticancer Research (2007), 27

The viability, cytolysis and apoptosis-mediated cellular death induced by giganteosides D and E (Gig-D and Gig-E) and hederacolchisides A and A1 (Hcol-A and Hcol- A1) were analysed in HL-60 cells ... [more ▼]

The viability, cytolysis and apoptosis-mediated cellular death induced by giganteosides D and E (Gig-D and Gig-E) and hederacolchisides A and A1 (Hcol-A and Hcol- A1) were analysed in HL-60 cells. Materials and Methods: the end-point metabolic (WST1) and lactate dehydrogenase (LDH) assays were used. Cell cycle analysis and apoptosis were measured by flow cytometry, DNA laddering and caspase 3 analyses. Results: the HL-60 cell line was more sensitive to Hcol-A1 and Gig-D (IC50 3-5 ÌM) than to Gig-E and Hcol-A (IC50 8-13 ÌM; WST1 assay). This was related to LDH release. The induction of apoptosis could be detected without caspase 3 activation after 24 h of treatment. DNA fragmentation could be detected only with Gig-D. With Hcol- A1 and Gig-D, an accumulation of cells in the S-phase and an increase of cells in sub-G1 peak were observed. By the annexinV-fluorescein isothiocyanate (FITC)/7-aminoactinomycin D (AAD) assay, the majority of cells were in late apoptosis with Gig-D, and in necrosis with Hcol-A1. Conclusion: Hcol-A1 is more cytotoxic than Gig-D, followed by Gig-E and finally Hcol-A. This is related to a membrane permeabilization effect, leading to cytolysis [less ▲]

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See detailGlobal strategy for the development of estrogenic compounds detection screening test
Collodoro, Mike ULg; Makasinga, Elu; Lemaire, Pascale ULg et al

Poster (2007, May)

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See detailMitoproteome plasticity of rat brown adipocytes in response to cold acclimation
Navet, Rachel ULg; Mathy, Grégory ULg; Douette, Pierre ULg et al

in Journal of Proteome Research (2007), 6(1), 25-33

Cold acclimation induces an adaptative increase in respiration in brown adipose tissue (BAT). A comparative analysis by two-dimensional differential in-gel electrophoresis of mitochondrial protein ... [more ▼]

Cold acclimation induces an adaptative increase in respiration in brown adipose tissue (BAT). A comparative analysis by two-dimensional differential in-gel electrophoresis of mitochondrial protein patterns found in rat control and cold-acclimated BAT was performed. A total of 58 proteins exhibiting significant differences in their abundance was unambiguously identified. Proteins implicated in the major catabolic pathways were up-regulated as were ATP synthase and mitofilin. Moreover, these results support the fact that adipocytes can balance their ATP synthesis and their heat production linked to UCP1-sustained uncoupling. [less ▲]

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