Laboratory of Mass Spectrometry

Le Touquet – Paris-Plage, France

Laboratory of Mass Spectrometry

Le Touquet – Paris-Plage, France

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Basso A.M.M.,University of Brasilia | Basso A.M.M.,Laboratory of Plant Pest Interaction | Pelegrini P.B.,Laboratory of Plant Pest Interaction | Mulinari F.,Laboratory of Plant Pest Interaction | And 6 more authors.
AMB Express | Year: 2015

In Brazil, there is a growing demand for specialised pharmaceuticals, and the high cost of their importation results in increasing costs, reaching US$ 1.34 billion in 2012 and US$ 1.61 billion in 2013. Worldwide expenses related to drugs could reach US$ 1.3 trillion in 2018, especially due to new treatments for hepatitis C and cancer. Specialised or high-cost pharmaceutical drugs used for the treatment of viral hepatitis, multiple sclerosis, HIV and diabetes are distributed free of charge by the Brazilian government. The glucagon peptide was included in this group of high-cost biopharmaceuticals in 2008. Although its main application is the treatment of hypoglycaemia in diabetic patients, it can also be used with patients in an alcoholic coma, for those patients with biliary tract pain, and as a bronchodilator. Therefore, in order to reduce biopharmaceutical production costs, the Brazilian government passed laws focusing on the development and increase of a National Pharmaceutical Industrial Centre, including the demand for the national production of glucagon. For that reason and given the importance and high cost of recombinant glucagon, the purpose of this study was to develop methods to improve production, purification and performance of the biological activity of recombinant glucagon. Glucagon was recombined into a plasmid vector containing a Glutathione S-transferase tag, and the peptide was expressed in a heterologous Escherichia coli system. After purification procedures and molecular analyses, the biological activity of this recombinant glucagon was examined using in vivo assays and showed a highly significant (p < 0.00001) and prolonged effect on glucose levels when compared with the standard glucagon. The experimental procedure described here facilitates the high level production of recombinant glucagon with an extended biological activity. © 2015, Basso et al.; licensee Springer.


Almonacid M.,University Pierre and Marie Curie | Almonacid M.,French National Center for Scientific Research | Celton-Morizur S.,University Pierre and Marie Curie | Celton-Morizur S.,French National Center for Scientific Research | And 13 more authors.
Current Biology | Year: 2011

In eukaryotes, cytokinesis generally involves an actomyosin ring, the contraction of which promotes daughter cell segregation. Assembly of the contractile ring is tightly controlled in space and time [1-4]. In the fission yeast, contractile ring components are first organized by the anillin-like protein Mid1 into medial cortical nodes [5-9]. These nodes then coalesce laterally into a functional contractile ring [10-13]. Although Mid1 is present at the medial cortex throughout G2 [14], recruitment of contractile ring components to nodes starts only at mitotic onset [12], indicating that this event is cell-cycle regulated. Polo kinases are key temporal coordinators of mitosis and cytokinesis [1], and the Polo-like kinase Plo1 is known to activate Mid1 nuclear export at mitotic onset [15, 16], coupling division plane specification to nuclear position [7]. Here we provide evidence that Plo1 also triggers the recruitment of contractile ring components into medial cortical nodes. Plo1 binds at least two independent sites on Mid1, including a consensus site phosphorylated by Cdc2. Plo1 phosphorylates several residues within the first 100 amino acids of Mid1, which directly interact with the IQGAP Rng2 [17], and influences the timing of myosin II recruitment. Plo1 thereby facilitates contractile ring assembly at mitotic onset. © 2011 Elsevier Ltd All rights reserved.


PubMed | Laboratory of Mass Spectrometry, Catholic University of Brasília, University of Brasilia and Laboratory of Plant Pest Interaction
Type: | Journal: AMB Express | Year: 2015

In Brazil, there is a growing demand for specialised pharmaceuticals, and the high cost of their importation results in increasing costs, reaching US$ 1.34 billion in 2012 and US$ 1.61 billion in 2013. Worldwide expenses related to drugs could reach US$ 1.3 trillion in 2018, especially due to new treatments for hepatitis C and cancer. Specialised or high-cost pharmaceutical drugs used for the treatment of viral hepatitis, multiple sclerosis, HIV and diabetes are distributed free of charge by the Brazilian government. The glucagon peptide was included in this group of high-cost biopharmaceuticals in 2008. Although its main application is the treatment of hypoglycaemia in diabetic patients, it can also be used with patients in an alcoholic coma, for those patients with biliary tract pain, and as a bronchodilator. Therefore, in order to reduce biopharmaceutical production costs, the Brazilian government passed laws focusing on the development and increase of a National Pharmaceutical Industrial Centre, including the demand for the national production of glucagon. For that reason and given the importance and high cost of recombinant glucagon, the purpose of this study was to develop methods to improve production, purification and performance of the biological activity of recombinant glucagon. Glucagon was recombined into a plasmid vector containing a Glutathione S-transferase tag, and the peptide was expressed in a heterologous Escherichia coli system. After purification procedures and molecular analyses, the biological activity of this recombinant glucagon was examined using in vivo assays and showed a highly significant (p<0.00001) and prolonged effect on glucose levels when compared with the standard glucagon. The experimental procedure described here facilitates the high level production of recombinant glucagon with an extended biological activity.


Carvalho J.O.,University of Sao Paulo | Silva L.P.,Laboratory of Mass Spectrometry | Sartori R.,University of Sao Paulo | Dode M.A.N.,Laboratory of Animal Reproduction | Dode M.A.N.,University of Brasilia
PLoS ONE | Year: 2013

Sperm dimensions and the question of whether X and Y chromosome-bearing sperm differ in size or shape has been of great interest, especially for the development of alternative methods to sort or classify sperm cells. The aim of the present study was to evaluate possible differences in the shape and size of the sperm head between X and Y chromosome-bearing sperm by atomic force microscopy (AFM). One ejaculate per bull (n = 4) was used. Each ejaculate was separated into four fractions: non-sexed (NS), sexed for X-sperm (SX), sexed for Y-sperm (SY) and a pooling of SX and SY samples (SXY). Using AFM, 400 sperm heads per group were measured. Twenty three structural features were assessed including one-, two- and three-dimensional parameters and shape descriptors. These measurements determine the micro- to nanoscale features of X- and Y-bearing chromosomes in sperm cells. No differences were observed for any individual variables between SX and SY groups. Next, a simultaneous evaluation of all features using statistical discriminant analysis was performed to determine if it was possible to distinguish to which group belong each individual cells. This analysis clearly showed, a distinct separation of NS, SXY, SX and SY groups. The recognition of this structural possibility to distinguish between X and Y sperm cell might improve the understanding of sperm cells biology. These results indicated that the associations of several structural measurements of the sperm cell head are promising candidates for development of a new method of sperm sexing. © 2013 Carvalho et al.


Quesada-Calvo F.,University of Liège | Bertrand V.,Mammalian Cell Culture Laboratory | Longuespee R.,Laboratory of Mass Spectrometry | Delga A.,University of Liège | And 9 more authors.
Journal of Proteomics | Year: 2015

Formalin-fixed paraffin-embedded (FFPE) specimens of patients are useful sources of materials for clinical research and have recently gained interest for use in the discovery of clinical proteomic biomarkers. However, the critical step in this field is the ability to obtain an efficient and repeatable extraction using the limited quantities of material available for research in hospital biobanks. This work describes the evaluation of the peptide/protein extraction using FFPE sections treated by the following two methods before shotgun proteomic analysis: a commercial solution (FFPE-FASP) (filter aided sample preparation) and an antigen retrieval-derived protocol (On Slice AR). Their efficiencies and repeatabilities are compared using data-independent differential quantitative label-free analysis. FFPE-FASP was shown to be globally better both qualitatively and quantitatively than On Slice AR. FFPE-FASP was tested on several samples, and differential analysis was used to compare the tissues of diverticulitis patients (healthy and inflammatory tissues). In this differential proteomic analysis using retrospective clinical FFPE material, FFPE-FASP was reproducible and provided a high number of confident protein identifications, highlighting potential protein biomarkers. Biological Significance: In clinical proteomics, FFPE is an important resource for retrospective analysis and for the discovery of biomarkers. The challenge for FFPE shotgun proteomic analysis is preparation by an efficient and reproducible protocol, which includes protein extraction and digestion. In this study, we analyzed two different methods and evaluated their repeatabilities and efficiencies. We illustrated the reproducibility of the most efficient method, FFPE-FASP, by a pilot study on diverticulitis tissue and on FFPE samples amount accessible in hospital biobanks. These data showed that FFPE is suitable for use in clinical proteomics, especially when the FFPE-FASP method is combined with label-free shotgun proteomics as described in the workflow presented in this work. © 2014 Elsevier B.V..


Turtoi A.,University of Liège | Turtoi A.,Laboratory of Mass Spectrometry | Blomme A.,University of Liège | Bellahcene A.,University of Liège | And 9 more authors.
Cancer Research | Year: 2013

Myoferlin is a member of the ferlin family of proteins that participate in plasma membrane fusion, repair, and endocytosis. While some reports have implicated myoferlin in cancer, the extent of its expression in and contributions to cancer are not well established. In this study, we show that myoferlin is overexpressed in human breast cancers and that it has a critical role in controlling degradation of the epidermal growth factor (EGF) receptor (EGFR) after its activation and internalization in breast cancer cells. Myoferlin depletion blocked EGF-induced cell migration and epithelial-to- mesenchymal transition. Both effects were induced as a result of impaired degradation of phosphorylated EGFR via dysfunctional plasma membrane caveolae and alteration of caveolin homo-oligomerization. In parallel, myoferlin depletion reduced tumor development in a chicken chorioallantoic membrane xenograft model of human breast cancer. Considering the therapeutic significance of EGFR targeting, our findings identify myoferlin as a novel candidate function to target for future drug development. ©2013 AACR.


Dos Anjos L.C.,University of Brasilia | Gomes F.M.M.,University of Brasilia | Do Couto L.L.,University of Brasilia | Mourao C.A.,University of Brasilia | And 4 more authors.
Life Sciences | Year: 2016

Anxiety disorders are major health problems in terms of costs stemming from sick leave, disabilities, healthcare and premature mortality. Despite the availability of classic anxiolytics, some anxiety disorders are still resistant to treatment, with higher rates of adverse effects. In this respect, several toxins isolated from arthropod venoms are useful in identifying new compounds to treat neurological disorders, particularly pathological anxiety. Thus, the aims of this study were to identify and characterize an anxiolytic peptide isolated from the venom of the social wasp Polybia paulista. The peptide was identified as Polisteskinin R, with nominal molecular mass [M + H]+ = 1301 Da and primary structure consisting of Ala-Arg-Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH. The anxiolytic effect was tested using the elevated plus maze test. Moreover, adverse effects on the spontaneous behavior and motor coordination of animals were assessed using the open field and rotarod tests. Polisteskinin R induced a dose-dependent anxiolytic effect. Animals treated with the peptide and diazepam spent significantly more time into the open arms when compared to the groups treated with the vehicle and pentylenetetrazole. No significant differences in spontaneous behavior or motor coordination were observed between the groups, showing that the peptide was well tolerated. The interaction by agonists in both known BK receptors induces a variability of physiological effects; Polisteskinin R can act on these receptors, inducing modulatory activity and thus, attenuating anxiety behaviors. The results of this study demonstrated that the compound Polisteskinin R exerted potent anxiolytic effects and its analogues are promising candidates for experimental pharmacology. © 2016 Elsevier Inc.


Barbosa E.F.,Laboratory of Mass Spectrometry | Barbosa E.F.,University of Brasilia | Monge-Fuentes V.,University of Brasilia | Oliveira N.B.,University of Brasilia | And 5 more authors.
IET Nanobiotechnology | Year: 2014

Brosimum gaudichaudii Tréc. (Moraceae) is a common Brazilian Cerrado plant known by its pharmaceutical industry relevance. The authors investigated the latex protein components and potential biotechnological applications. Some protein fragments had their sequences elucidated, presenting similarities to jacalin and Kunitz-type trypsin inhibitors. Amino acid residue modifications were found, such as glutamine N-terminal residue cyclisation into pyroglutamic acid residue, and mass differences corresponding to hexoses and N-acetylhexosamine presence. The latex was used to produce a nanoscale structured film, which presented an increased attraction and reduced adhesion behaviours. The film presented high homogeneity, as observed by low nanoroughness values, probably because of its intrinsic components, such as the jacalin-like protein that has known agglutination properties. The immobilised Kunitz-type trypsin inhibitor presence in the latex film allow us to point out to applications related to this inhibition, as in active food packaging, since these peptidase inhibitors are able to inhibit pests and microorganism proliferation. © The Institution of Engineering and Technology 2014.


Baiwir D.,University of Liège | Mazzucchelli G.,Laboratory of Mass Spectrometry | Smargiasso N.,Laboratory of Mass Spectrometry | Quesada-Calvo F.,University of Liège | And 4 more authors.
EuPA Open Proteomics | Year: 2015

Off-line sample prefractionations applied prior to biomarker discovery proteomics are options to enable more protein identifications and detect low-abundance proteins. This work compared five commercial methods efficiency to raw serum analysis using label-free proteomics. The variability of the protein quantities determined for each process was similar to the unprefractionated serum. A 49% increase in protein identifications and 12.2% of reliable quantification were obtained. A 61 times lower limit of protein quantitation was reached compared to protein concentrations observed in raw serum. The concentrations of detected proteins were confronted to estimated reference values. © 2015 The Authors.


von Gal Milanezi N.,University of Brasilia | Mendoza D.P.G.,University of Brasilia | de Siqueira F.G.,University of Brasilia | Silva L.P.,Laboratory of Mass Spectrometry | And 2 more authors.
Bioenergy Research | Year: 2012

Aspergillus niger van Tieghem LPM 93 was shown in an earlier study to produce the most thermostable β-xylanase, which was effective for improving brightness and delignification of non-delignified and oxygen-bleached samples of eucalyptus kraft pulp. Here, we report the production, purification, and characterization of a xylan-degrading enzyme (XynI) from this strain grown in submerged liquid cultivation on medium containing sugar cane bagasse as the carbon source. XynI was isolated by ultrafiltration and gel-filtration chromatography and characterized. The fungus displayed high levels of xylanolytic activity after the second day of cultivation, and this activity remained constant up to the 50th day. The molecular mass of XynI was in the range of 32-33 kDa as determined by mass spectrometry and SDS-PAGE. The two-dimensional gel electrophoresis analysis showed the existence of multiple forms of β-xylanases in XynI. XynI showed the highest activity at 50°C and pH 4.5 and was stable in sodium acetate buffer at pH 4.5. The K m and V max values were 47.08 mg/ml and 3.02 IU/ml, respectively. Salts inhibited the activity of XynI to different degrees. N-Bromosuccinimide caused marked inhibition of XynI. On the other hand, β-mercaptoethanol and l-tryptophan were the best enzyme activators. © 2011 Springer Science+Business Media, LLC.

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