Analytical Biochemistry and Proteomics Unit

>>Analytical Biochemistry and Proteomics Unit

The Analytical Biochemistry and Proteomics Unit (Ubypa) —an IP Montevideo and Instituto de Investigaciones Biológicas Clemente Estable (IIBCE) Mixed Unit— has the objective of carrying out and supporting biomedical research projects based on mass spectrometry (MS) and proteomics. It also offers training, scientific assistance and access to EM-based proteomic technologies to the local scientific community; and contributes to local and regional education programs in this area.

During the last years, the Unit incorporated mass spectrometers and developed the know-how to expand the quality and type of analytical procedures available. Currently, our analytical portfolio includes “shotgun” proteomic strategies, as well as gel-based strategies; in vivo and in vitro interactome studies; and analysis of post-translational protein modifications.

The research projects of our group focus on the study of signaling mechanisms in mycobacteria using proteomic approaches, with emphasis on the analysis of protein phosphorylation. In particular, we are interested in understanding some key processes for pathogenic mycobacteria, such as Mycobacterium tuberculosis, which are related to their ability to survive inside the host.


Magdalena Portela

Technical Assistant

madelon@pasteur.edu.uy


Analía Lima

Technical Assistant, PhD student

alima@pasteur.edu.uy


Jessica Rossello

PhD student

jrossello@pasteur.edu.uy


MSc Bernardina Rivera

Technical Assistant

brivera@pasteur.edu.uy


Alejandro Leyva

Technical Assistant, PhD student

alejandrolp@pasteur.edu.uy

  • The role of protein phosphorylation in the regulation of biological processes in Mycobacterium tuberculosis.
    Our work focuses on the characterization of signaling pathways mediated by phosphorylation in mycobacteria. In the past, we described the regulatory mechanisms of Ser/Thr kinases and identified some of their substrates and targets “downstream” in the signaling pathways. At present, we are using in vivo cross-linking in combination with mass spectrometry to obtain a “snapshot” of the protein-protein interactions in the living bacteria, with emphasis in the study of the interactome of the previously identified kinase-substrates. This has allowed us to begin to elucidate phosphorylation-dependent interactions that participate in the regulation of nitrogen uptake and cell division.

  • HPLC, Agilent 1200

  • Capilar HPLC, Agilent 1200;

  • Nano HPLC, Easy-nLC 1000, Thermo

  • Nano HPLC Ultimate 300, Thermo

  • 2D Electrophoresis, EttanIPGphor + EttanDaltSix

  • Typhoon FLA 9500, GE Healthcare

  • 4800 MALDI TOF/TOF Mass Spectrometer, Abi Sciex

  • LTQ Velos + ETD Mass Spectrometer, Thermo

  • Q-exactive (Q-Orbitrap), Thermo

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.

To perform a routine analysis, please contact the technical team to confirm availability to the email ubypa@pasteur.edu.uy . When your application has been accepted, we will contact you to schedule the analysis.

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.

  • “Shotgun” based proteomics.

  • 2-D gel electrophoresis based proteomics.

  • Quantitative proteomics.

  • Post-translational modification analysis.

  • De novo peptide sequencing.

For scientific inquiries, please write to ubypa@pasteur.edu.uy . We will try to be flexibile to find the best way to use our facilities.

  • ICGEB & UNU-BIOLAC “Proteome Analysis by Mass Spectrometry”. October 15-23, 2018. Organizers: Rosario Durán, Paulo Carvalho & Carlos Batthyány.

  • UNU-BIOLAC & PEDECIBA “Proteome Analysis by Mass Spectrometry”; Nov28-Dec2, 2016.

  • Organizers: Rosario Durán, Paulo Carvalho & Carlos Batthyány.

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

  • UNU-Biolac- “Mass Spectrometry (MS) in Proteomics” Institut Pasteur de Montevideo –Nov26-Dec8, 2012. Organizers: C. Batthyany, R. Durán

  • EMBO World Course – “Mass Spectrometry in Protein Analysis and Characterization”, Institut Pasteur de Montevideo, Uruguay, March 16-26, 2010. Organizers: C. Cerveñansky, R. Durán & C. Batthyany

  • “Workshop on mass spectrometry and its application on protein analysis”, Programa de Pós-Graduação em Bioquímica Faculdade de CiênciasFarmacêuticas de Ribeirão Preto Universidade de, São Paulo, Brasil. October 19-24, 2009. Organizadores: R. Durán & C. Batthyany.

  • Course “Integrating IP Montevideo technologies”. Module: “Protein Characterization using Mass Spectrometry”. October 6, 2017. Organizers: A. Lima and M. Portela.

  • 2018-2022 – Molecular mechanisms of phospho-dependent regulation and assembly of the bacterial divisome. ANR Francia. Coordinator P. Alzari, Institut Pasteur, Paris

  • 2015-2018 – FHA domain mediated signaling cascades in mycobacteria and its role in the adaptation to the host environment. Responsible: Rosario Durán. Fondo Clemente Estable, Modalidad I. FCE_1_2014_1_104045.

  • 2012-2014 – Exploring the role of mosquito’s saliva in the transmission of Rift Valley fever; Actions Concertées Interpasteuriennes (ACIP). Coordinador: V. CHOUMET (Paris). Responsables en Uruguay: C. Batthyány & R. Durán.

  • 2014-2016 – Comparative proteomic analysis of two Pseudomonas aeruginosa strains showing differential adhesivity to epithelial cells. Responsible: J. Rossello. Fondo Clemente Estable, Modalidad II. FCE_3_2013_1_100344.

  • 2014-2016 – Towards the elucidation of the molecular mechanism used by PknG to exert its role as virulence factor. Responsible: M. Gil. Fondo Clemente Estable, Modalidad II. FCE_3_2013_1_100358.

  • Piñas GE, Reinoso-Vizcaino NM, Yandar Barahona NY, Cortes PR, Duran R, Badapanda C, Rathore A, Bichara DR, Cian MB, Olivero NB, Perez DR, Echenique J. 2018. Crosstalk between the serine/threonine kinase StkP and the response regulator ComE controls the stress response and intracellular survival of Streptococcus pneumoniae. PLoS Pathog. 14(6):e1007118

  • Fló M., Margenat M., Pellizza L., Graña M., Durán R., Báez A., Salceda E., Soto E., Alvarez B., Fernández C. 2017. Functional diversity of secreted cestodeKunitz proteins: Inhibition of serine peptidases and blockade of cation channels. PLoSPathogens. 13(2):e1006169

  • Prieto D., Sotelo N., Seija N., Sernbo S., Abreu C., Durán R., Gil M., Sicco E., Irigoin V., Oliver C., Landoni A.I., Gabus R., Dighiero G., Oppezzo P. 2017. S100-A9 protein in exosomes from chronic lymphocytic leukemia cells promotes NF-κB activity during disease progression. Blood. 130(6):777-788

  • Silva A.R.F., Lima D.B., Leyva A., Duran R., Batthyany C., Aquino P.F., Leal J.C., Rodriguez J.E., Domont G.B., Santos M.D.M., Chamot-Rooke J., Barbosa V.C., Carvalho P.C. 2017 DiagnoProt: a tool for discovery of new molecules by mass spectrometry. Bioinformatics. 33(12):1883-1885. doi: 10.1093/bioinformatics/btx093

  • Silva A.R.F., Lima D.B., Leyva A., Duran R., Batthyany C., Aquino P.F., Leal J.C., Rodriguez J.E., Domont G.B., Santos M.D.M., Chamot-Rooke J., Barbosa V.C., Carvalho P.C. 2017 DiagnoProt: a tool for discovery of new molecules by mass spectrometry. Bioinformatics. 33(12):1883-1885. doi: 10.1093/bioinformatics/btx093

  • Cabrera G., Lundberg U., Rodríguez-Ulloa A., Herrera M., Machado W., Portela M., Palomares S., Espinosa L.A., Ramos Y., Durán R., Besada V., Vonasek E., González L.J. 2017 Protein content of the Hylesiametabus egg nest setae (Cramer [1775]) (Lepidoptera: Saturniidae) and its association with the parental investment for the reproductive success and lepidopterism. J Proteomics. 150:183-200

  • Folle A.M., Kitano E.S., Lima A., Gil M., Cucher M.; Mourglia-Ettlin G., Iwai L.K., Rosenzvit M., Battyány C., Ferreira A.M. Characterisation of Antigen B protein species present in the hydatid cyst fluid of Echinococcuscanadensis G7 genotype. PLoS Neglected Tropical Diseases, 2017.

  • Rossello J., Lima A., Gil M., Duarte J.R., Correa A., Carvalho P.C., Kierbel A., Durán R. 2017. The EAL-domain protein FcsR regulates flagella, chemotaxis and type III secretion system in Pseudomonas aeruginosa by a phosphodiesterase independent mechanism. ScientificReports. 7(1): 10281.

  • Gil M, Graña M, Schopfer FJ, Wagner T, Denicola A, Freeman BA, Alzari PM, Batthyány C, Durán R. Inhibition of Mycobacterium tuberculosis PknG by non-catalytic rubredoxin domain specific modification: reaction of an electrophilic nitro-fatty acid with the Fe-S center. Free Radic. Biol. Med. 65, 150–161 (2013).

  • O’Hare, H; Durán, R; Cerveñansky,C, Bellinzoni, M.; Wehenkel, A.; Pritsch, O.; Obal, G; Baumgartner, J; Johnsson,K; Alzari, PM. Regulation of glutamate metabolism by proteinkinases in mycobacteria. Molecular Microbiology 70, 1408-1423 (2008).

  • Durán, R., Villarino, A., Bellinzoni, M., Wehenkel, A., Fernandez, P., Boitel, B., Cole, S.T., Alzari, P.M., Cervenansky, C. Conserved autophosphorylation pattern in activation loops and juxtamembrane regions of Mycobacterium tuberculosis Ser/Thr protein kinases. Biochem. Biophys. Res. Commun. 333:858-67 (2005).

  • Villarino*, A., Durán*, R., Wehenkel, A., Fernandez, P., England, P., Brodin, P., Cole, S.T., Zimny-Arndt, U., Jungblut, P.R., Cervenansky, C., Alzari, P.M. Proteomic identification of M. tuberculosis protein kinase substrates: PknB recruits GarA, a FHA domain-containing protein, through activation loop-mediated interactions. J. Mol. Biol. 350:953-63 (2005). * These authors have equally contributed to the work.