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Analytical Biochemistry and Proteomics Unit

 

MEMBERS

  • Rosario Durán, PhD (Interim Head, Investigator IIBCE – IP Montevideo)
  • Carlos Batthyány, MD, PhD (Investigator-IP Montevideo; Adjunct Professor of Biochemistry, School of Medicine, UdelaR)
  • Horacio Botti, MD, PhD (Adjunct Investigator, IP Montevideo)
  • Magdalena Portela (Technical Assistant – School of Sciences/IP Montevideo)
  • Analía Lima, MSc. (Technical Assistant, PhD student)
  • Magdalena Gil, Biochemist (Technical Assistant, PhD student; ANII Fellow)
  • Jessica Rossello, Biochemist (Technician, PhD student; ANII Fellow)
  • Bernardina Rivera, Biochemist (Technician, Graduate student)

Associate Members

  • María Noel Álvarez, PhD (Associate Investigator, Adjunct Professor of Biochemistry, School of Medicine, UdelaR, Uruguay)
  • Leonel Malacrida, PhD (Associate Investigator, Assistant Profesor, Pathophysiology Department, School of Medicine, UdelaR, Uruguay)
  • Virginia López, PhD (Adjunct Professor of Organic Chemistry, School of Chemistry and Science, UdelaR)

Students

  • Jorge Rodriguez, Biochemist (PhD student; ANII Fellow)
  • Gonzalo Spera, M.D. (MSc student)
  • Adriana Carlomagno, M.D. (MSc student)
  • Rosina Dapueto, M.Sc. (PhD Student, ANII Fellow)
  • Germán Galliusi (MSc student, ANII Fellow)
  • Rosina Toledo (MSc student, CSIC)
  • Josefina Peña (Undergraduate student, ANII Fellow)

MAIN EQUIPMENT

services

SERVICES

Our Unit received in January 2007 a MALDI TOF-TOF MS instrument (AB-SCIEX, Framingham, USA) and, in December 2009, completed the MS platform with the arrival of a nano-electrospray/ion trap LTQ Velos instrument (Thermo, USA). Both instruments complement each other and expand the quality and type of mass analytical procedures we can offer to local and regional research groups.

There are two general modes to get access to the Unit facilities:

Routine Service

For routine analysis, users are welcome to access the UByPA as a “fee for service facility” supported by the Institut Pasteur de Montevideo. The facility offers this kind of service to researchers in the region, with priority given to users from the Institute and local academy. The analysis will be performed by members of our technical staff and will be done following standard protocols. The routine analysis includes analysis and interpretation of raw data based on routine practices only.

Routine analysis includes:

  • 2-D gel electrophoresis.
  • Protein sample preparation for MS analysis: in-gel digestion, in-solution digestion, desalting.
  • Molecular mass determination for peptides and small proteins by MS.
  • Protein identification by MALDI-TOF/TOF MS (peptide mass fingerprinting, MS/MS ion search) and database search.

In 2013 we analyzed 1178 different samples from 83 different groups: 55% of the samples were from our local academy, 42% were from the regional academy (Argentine, Brazil, Chile, Colombia, and Venezuela) and 3% from private industry.

In 2014 we analyzed 1238 samples from different groups from local and regional academy.

Non-Routine Service

Collaborative research projects, beyond routine services, are welcome. Members of the Unit are expected to significantly contribute to the conception, design of experiments and custom-design protocols, original ideas as well as data analysis and interpretation beyond routine practice.

Non routine analysis includes:

  • Custom sample preparation.
  • Post-translational modification analysis.
  • 2-D gel electrophoresis based proteomics.
  • “Shotgun” based proteomics.
  • Quantitative proteomics.
  • De novo peptide sequencing.
  • Glycomics and glycoproteomics.

Forms:

  1. Determinación de masa molecular de péptidos y proteínas pequeñas por MS
  2. Electroforesis Bidimensional
  3. Identifcación de proteínas por MALDI-TOF MS/MS

PRICES

Prices for routine services (american dollar, USD)

Científico Nacional Científico Regional* Científico InternacionalUsuarios Extra-académicos
Determinación de masa molecular 12 36 72
Gel 2D 10*10 cm + tinción 60 240 480
Gel 2D 20*20 cm + tinción consultar consultar consultar
2D Clean Up Kit para purificación 12 48 96
Cuantificación con 2D Quant Kit 8 36 72
Identificación de proteínas por MALDI-TOF/TOF MS 24 60 165
  • *Latin America

 

RESEARCH

In the past years, members of our group have been involved in different areas of biological/biochemical research. A major contribution made by the UByPA´s scientist was the incorporation of modern mass spectrometry (MS) and 2D-DIGE to our local academy, bringing totally new analytical capabilities to perform comprehensive protein studies, including posttranslational modifications of proteins and the ability to decode cell signaling networks.Nowadays, we are involved in three main areas of research concerning protein-mediated cell signaling events:

CELL SIGNALING PATHWAYS IN PATHOGENIC BACTERIA: A PROTEOMIC APPROACH

  • Characterization of Mycobacterium tuberculosis Ser/Thr kinase PknG signaling pathways. PI: Rosario Durán; Graduate Thesis Project of M. Gil, PEDECIBA, UdelaR, 2011-to date.
  • Effects of Mycobacterium tuberculosis Ser/Thr kinase PknG on the macrophage. PIs: Rosario Durán, Carlos Batthyány and MN Álvarez; Graduate Thesis Project of MSc A.Lima, Pro.In.Bio., UdelaR, 2011-to date.
  • Proteomic characterization of c-di-GMP mediated transition from a planktonic to surface-associated Pseudomonas aeruginosa. PIs: R. Durán; Graduate Thesis Project of J. Rossello, PEDECIBA, UdelaR, 2013 to date).

BIOLOGICAL EFFECTS OF SECOND GENERATION NITROALKENE TOCOPHEROL ANALOGS:

Atherosclerosis is an epidemic worldwide disease and leading cause of death in developed countries. Two main pathogenic events are well recognized in the generation of the atheroma plaques: inflammation and lipid accumulation via LDL deposition/foam cells formation. Inflammation is a key event mediated by inflammasome activation by sterile signals (i.e. cholesterol crystals) and precedes massive lipid accumulation.

In our work we envision a new pharmacological strategy for the treatment and prevention of atherosclerosis. We designed a hybrid compound analog of -tocopherol to which we added the electrophilic nitroalkene group (1). The rationale for our idea is that the nitroalkene-tocopherol analog will be selectively incorporated into the lipoprotein particles during their normal metabolism due to the presence of the chromanol structure. Once incorporated, lipoproteins will transport the compound through the body, including to the atherosclerotic lesions, were it may exert the potent anti-inflammatory and anti-atherogenic properties of the nitroalkenes.

We synthesized two series of tocopherol analogs and performed their physicochemical and biological characterization. Nitroalkene-tocopherol analogs are electrophiles that are being incorporated into lipoproteins both in vitro and in vivo and exhibit potent anti-inflammatory activity: A- inhibit pro-inflammatory cytokines (IL-6, MCP-1, TNF) secretion controlled by NFB factor & B- induce expression of phase 2 enzymes regulated by Nrf2/Keap-1 (HO-1, GCLM, NQO1) in murine macrophages; C- inhibit interleukin 1- secretion by NLRP3 inflammasome in THP-1 cells. We are now evaluating their capabilities to inhibit the development of atherosclerotic plaques formation in two animal models: apo E-/- and zebrafish.

    1. Design and Development of Nitroalkene Tocopherol and Analogs for Use in the Treatment and Prevention of Inflammatory Mediated Diseases. PIs: C. Batthyány and V. López; Graduate Thesis Project of J. Rodriguez, PEDECIBA, UdelaR, 2012 to date).
    2. Electrophilic mediated protein modifications.
      1. “Composition and Method for Inhibition of PknG from Mycobacterium tuberculosis”, PIs C. Batthyány & R. Durán (U.S. PCT Application No. 61/835,416; 2014).
      2. Molecular Mechanisms of Nitroalkene mediated anti-inflammatory cell signaling events; PIs C. Batthyány & H. Botti.

HYDRATION AND SUPRAMOLECULAR ORGANIZATION OF CELLULAR MEMBRANES

  1. “Hydration and Supramolecular Organization Studies of Lamellar Bodies in A549 Lung Cells using LAURDAN fluorescence”; PI L. Malacrida.
  2. Purification and molecular characterization of lung surfactant from different species: SP-B, SP-C and lipids fractions; PI L. Malacrida.
  3. Effect of tocopherol in cellular membranes; PIs L. Malacrida & C. Batthyány.
  4. Characterization of phagolysosome maturation by fluorescence microscopy approaches; PIs Leonel Malacrida & R. Durán.

EDUCATION-COURSES

ORGANIZATION OF COURSES

  1. “Proteome Analysis by Mass Spectrometry”; UNU-BIOLAC & RIIP (Institut Pasteur International Network). September 1 to 12, 2014. Organizers: Rosario Durán & C. Batthyány.

TRAINING OF STUDENTS

Graduate students:

  • Analía Lima. Pro.In.Bio. “Caracterización molecular del proceso de inhibición del fagosoma por una quinasa de Mycobacterium tuberculosis”. Directores Académicos: C. Batthyány, R. Durán, MN. Álvarez.
  • Magdalena Gil. PEDECIBA Química, “Regulación de la actividad quinasa de PknG de M. tuberculosis y su rol en las primeras etapas de la infección”. Director: A. Denicola; Co-Director: R. Durán.
  • Jorge Rodríguez. PEDECIBA Química. “I+D de análogos de la vitamina E liberadores de óxido nítrico o nitroalquenos como potenciales fármacos para prevención primaria de aterosclerosis”. Director: V. López. Co-director. C. Batthyany.
  • Rosina Dapueto. Pro.In.Bio. “Inhibidores de CD38 y nitroalquenos derivados de la vitamina E para el tratamiento de enfermedades cardiovasculares”. Director Académico Dr. Carlos Batthyány; Co-dirección: Drs. Virginia López y Carlos Escande.

Magister in Science students:

  • Jessica Rossello. PEDECIBA Biología. “Estudio de la adhesión y agregación de Pseudomonas aeruginosa en células epiteliales mediante aproximaciones proteómicas” Director: R. Durán Co-Director: A. Kierbel.
  • Dr. Gonzalo Spera. Pro.In.Bio. “Proteómica diferencial de líneas celulares de cáncer de mama metástasico HER2 negativo sensibles y resistentes a Docetaxel”. Director: Dr. C. Batthyány, Co-Directors: Dra. C. Touriño, Dra. L. Delgado.
  • Bernadina Rivera. PEDECIBA Química. “Vías de señalización mediadas por PknG y su regulación en micobacterias”. Director: Dra R. Durán, Co-Director: Dr. C. Batthyány.

Undergraduate students:

  • Josefina Peña. Lic. Bioquímica. “Modulación del proteoma del fagosoma por una quinasa de Mycobacterium tuberuculosis“. Initiation into Research Fellowship ANII. Director: R. Durán; Co-Director: A. Lima
  • Germán Galliusi. Lic. de Bioquímica. “Mecanismos anti-inflamatorios de los nitroalquenos: Aparente contra regulación del Inflamasoma NLRP3 y la Apoptosis”. Initiation into Research Fellowship ANII. Director: C. Batthyány; Co-Director: H. Botti.
  • Rosina Toledo Gallo. Lic. Bioquímica. “Purificación Analítica y Preparativa de las Proteínas hidrofóbicas del Surfactante Pulmonar”. Initiation into Research Fellowship ANII.

Training stages at the Unit:

  • Lorena Pardo, MD. (Graduate student, Microbiology Department, School of Medicine, UdelaR); Oct 2013 to date.
  • Germán Rosano, PhD. (Instituto de Biología Molecular y Celular de Rosario, Argentina) Financial support: CONICET, September, 2014.
  • Luciana Fleitas, Graduate Student. (Facultad de Ciencias, UdelaR). April-May, 2014
  • Paola Scavone, PhD. (Instituto de Investigaciones Biológicas Clemente Estable). March-April, 2014.
  • Adriana Martínez, Graduate Student, February 2014 – to date.

GRANTS

  1. “Development of a novel class of anti-atherogenic agents: electrophilic nitroalkenes-Vitamin E (α-tocopherol) analogs”. (2013 – 2015); CABBIO; PI Carlos Batthyány; Amount Granted USD 30.000.
  2. “N‐Glycan fingerprint of exosomes in Chagas and Cancer diseases”. (2014); Interdisciplinary projects IP Montevideo; Coordinator C. Batthyány, Amount granted USD 20.000.
  3. Exploring the role of mosquito’s saliva in the transmission of Rift Valley fever” (2012-2014); Actions Concertées Interpasteuriennes (ACIP). Scientific coordinator: V. CHOUMET (Paris). Uruguayan PIs: C. Batthyány & R. Durán. Amount Granted: EUR 18.000.
  4. “Caracterización nutricional y de compuestos bioactivos del trigo en Uruguay. Variabilidad de genotipos y ambientes”; (2014 – 2016); FPTA – INIA: Contrato de Servicio PIs C. Batthyány & L. Malacrida; Amount Granted USD 32.000.
  5. “Identification of tumor associated antigens”; (2014 – 2015); Private Company: Contrato de Servicio; PIs R. Durán & C. Batthyány; Amount Granted: USD 35.500.
  6. Anti-atherogenic effects and molecular mechanisms of nitroalkene tocopherol analgos: a novel pharmacological approach” (2014-2016); PIs C. Batthyány & H. Botti; Amount Granted: USD 30.000.
  7. Análisis proteómico comparativo de dos cepas de P. aeruginosa con distinta capacidad de adhesión a células epiteliales” (2014-2016); J. Rossello (FCE_3_2013_1_100344); Amount Granted: USD 25.000.
  8. Hacia la elucidación del mecanismo molecular utilizado por PknG para ejercer su rol como factor de virulencia” (2014-2016). M. Gil (FCE_3_2013_1_100358); Amount Granted: USD 20.000.
  9. Surfactante Pulmonar durante la Lesión Pulmonar Aguda: Abordaje estructural, dinámico y funcional (2013 – 2015) CSIC I+D 2012, PI: L. Malacrida, Amount Granted: USD 38.000.

PUBLICATIONS

  1. Turell L, Botti H, Bonilla L, Torres MJ, Schopfer F, Freeman BA, Armas L, Ricciardi A, Alvarez B, Radi R (2014) HPLC separation of human serum albumin isoforms based on their isoelectric points. J Chromatogr B Analyt Technol Biomed Life Sci 944: 144-151.
  2. Trochine A, Alvarez G, Corre S, Faral-Tello P, Duran R, Batthyany C, Cerecetto H, Gonzalez M, Robello C (2014) Trypanosoma cruzi chemical proteomics using immobilized benznidazole. Exp Parasitol 140: 33-38.
  3. Trajtenberg F, Albanesi D, Ruetalo N, Botti H, Mechaly AE, Nieves M, Aguilar PS, Cybulski L, Larrieux N, de Mendoza D, Buschiazzo A (2014) Allosteric activation of bacterial response regulators: the role of the cognate histidine kinase beyond phosphorylation. MBio 5: e02105.
  4. Randall LM, Manta B, Hugo M, Gil M, Batthyany C, Trujillo M, Poole LB, Denicola A (2014) Nitration transforms a sensitive peroxiredoxin 2 into a more active and robust peroxidase. J Biol Chem 289: 15536-15543.
  5. Morero NR, Botti H, Nitta KR, Carrion F, Obal G, Picardeau M, Buschiazzo A (2014) HemR is an OmpR/PhoB-like response regulator from Leptospira, which simultaneously effects transcriptional activation and repression of key haem metabolism genes. Mol Microbiol 94: 340-352.
  6. Mon ML, Moyano RD, Viale MN, Colombatti Olivieri MA, Gamietea IJ, Montenegro VN, Alonso B, Santangelo Mde L, Singh M, Duran R, Romano MI (2014) Evaluation of cocktails with recombinant proteins of Mycobacterium bovis for a specific diagnosis of bovine tuberculosis. Biomed Res Int 2014: 140829.
  7. Martinez A, Peluffo G, Petruk AA, Hugo M, Pineyro D, Demicheli V, Moreno DM, Lima A, Batthyany C, Duran R, Robello C, Marti MA, Larrieux N, Buschiazzo A, Trujillo M, Radi R, Piacenza L (2014) Structural and Molecular Basis of the Peroxynitrite-mediated Nitration and Inactivation of Trypanosoma cruzi Iron-Superoxide Dismutases (Fe-SODs) A and B: Disparate Susceptibilities Due To The Repair Of Tyr35 Radical By Cys83 In Fe-SodB Through Intramolecular Electron Transfer. J Biol Chem 289: 12760-12778.
  8. Malacrida L, Reta G, Piriz H, Rocchiccioli F, Botti H, Denicola A, Briva A (2014) Sevoflurane anesthesia deteriorates pulmonary surfactant promoting alveolar collapse in male Sprague-Dawley rats. Pulm Pharmacol Ther 28: 122-129.
  9. Dieterle ME, Bowman C, Batthyany C, Lanzarotti E, Turjanski A, Hatfull G, Piuri M (2014) Exposing the secrets of two well-known Lactobacillus casei phages, J-1 and PL-1, by genomic and structural analysis. Appl Environ Microbiol 80: 7107-7121.

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