Analytical Biochemistry and Proteomics

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.

Members

Rosario Durán, PhD

Rosario Durán, PhD

Head

Magdalena Portel, BSc

Magdalena Portel, BSc

Senior associate technician

Analía Lima, PhD

Analía Lima, PhD

Senior associate researcher

Jessica Rossello, MSc

Jessica Rossello, MSc

Doctoral student

Bernardina Rivera, MSc

Bernardina Rivera, MSc

Associate researcher

Alejandro Leyva, BSc

Alejandro Leyva, BSc

Senior associate technician

Paulo Carvalho, PhD

Paulo Carvalho, PhD

Associate researcher - Fiocruz Paraná

Azalia Rodríguez, MSc

Azalia Rodríguez, MSc

Doctoral student

Flavio Pazos Obregón, PhD

Flavio Pazos Obregón, PhD

Senior associate technician

Research lines

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.

Main equipment

  • 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
  • Microfraction collector for MALDI plating, Probot LC-Packing

Services

ROUTINE ANALYSIS

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.

The forms are:

  1. Determination of molecular mass of peptides and small proteins by MS.
  2. 2-D gel electrophoresis.
  3. Protein identification by MALDI-TOF MS/MS.

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
  • Interactome analysis
  • 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.

Download the sample preparation guide here.

FAQs about routine analysis here.

Courses

  • 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”. November 28 – December 2, 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. November 26 – December 8, 2012. Organizers: C. Batthyany, R. Durán
  • EMBO World Practical Course. “Mass Spectrometry in Protein Analysis and Characterization”, EMBO, IPMONT, ANII; Montevideo, Uruguay. March 16-26, 2010. Organizers: C. Cerveñansky, R. Durán & C. Batthyany
  • “Workshop on mass spectrometry and it’s 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. Organizers: R. Durán & C. Batthyany.
  • Course “Integrating IP Montevideo technologies”. Module: “Protein Characterization using Mass Spectrometry”. October 6, 2017. Organizers: A. Lima and M. Portela.

Projects

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). Coordinator: V. CHOUMET (Paris). Responsibles in 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.

Main publications

vacio
2021
  • Lima A, Leyva A, Rivera B, Portela MM, Gil M, Cascioferro A, Lisa MN, Wehenkel A, Bellinzoni M, Carvalho PC, Batthyány C, Alvarez MN, Brosch R, Alzari PM, Durán R. (2021) Proteome remodeling in the Mycobacterium tuberculosis PknG knockout: Molecular evidence for the role of this kinase in cell envelope biogenesis and hypoxia response. Journal of Proteomics. 244, 104276 DOI: 10.1016/j.jprot.2021.104276
  • Lisa MN, Sogues A, Barilone N, BaumgartM3, Gil M, Graña M, Durán R, Biondi RM, Bellinzoni M, Bott M, Alzari PM (2021) A Tetratricopeptide Repeat Scaffold Couples Signal Detection to OdhI Phosphorylation in Metabolic Control by the Protein Kinase PknG. mBio e0171721. doi: 10.1128/mBio.01717-21.
  • Martinez-Sanguiné AY, D’Alessandro B, Langleib M, Traglia GM, Mónaco A, Durán R, Chabalgoity JA, Betancor L, Yim L. (2021) Salmonella enteric Serovars Dublin and Enteritidis Comparative Proteomics Reveals Differential Expression of Proteins Involved in Stress Resistance, Virulence, and Anaerobic Metabolism. Infect Immun. 16, 89(3). doi: 10.1128/IAI.00606-20.
  • Ott C, Tomasina F, Campolo N, Bartesaghi S, Mastrogiovanni M, Leyva A, Batthyany C, Meinl W, Grune T, Radi R. (2021) Decreased proteasomal cleavage at nitrotyrosine sites in proteins and peptides. Redox Biology. 46, 102106 DOI: 10.1016/j.redox.2021.102106
  • González LJ, Encinosa Guzmán PE, Machado W, Pousa S, Leyva A, Cano Arguelles AL, Cabrera G, Espinosa LA, Parra R, Hernández R, Bello Soto Y, Ledesma FL, Joglar M, Guirola O, Ulrich Kurt L, Carvalho PC, Cabrales A, Garay H, Besada V, Durán R, Takao T, Estrada MP, Rodríguez-Mallon A. (2021). Synthesis, LC-MS/MS analysis, and biological evaluation of two vaccine candidates against ticks based on the antigenic P0 peptide from R. sanguineus linked to the p64K carrier protein from Neisseria meningitidis. Anal Bioanal Chem. 413, 5885–5900 DOI: 10.1007/s00216-021-03569-0
  • Cardozo V, Vaamonde L, Parodi-Talice A, Zuluaga MJ, Agrati D, Portela M, Lima A, Blasina F, Dajas F, Bedó G. (2021) Multitarget neuroprotection by quercetin: Changes in gene expression in two perinatal asphyxia models. Neurochem Int. 147:105064. doi: 10.1016/j.neuint.2021.105064.
  • Chalar C, Clivio G, Montagne J, Costábile A, Lima A, Papa NG, Berois N, Arezo MJ. (2021) Embryonic developmental arrest in the annual killifish Austrolebias charrua: A proteomic approach to diapause III..PLoS One. 16(6):e0251820. doi: 10.1371/journal.pone.0251820.
  • Demicheli V, Tomasina F, Sastre S, Zeida A, Tórtora V, Lima A, Batthyány C, Radi R. (2021) Cardiolipin interactions with cytochrome c increase tyrosine nitration yields and site-specificity. Arch Biochem Biophys. 703:108824. doi: 10.1016/j.abb.2021.108824.
2020
  • Rivera B, Leyva A, Portela MM, Moratorio G, Moreno P, Durán R, Lima A. (2020) Quantitative proteomic dataset from oro- and naso-pharyngeal swabs used for COVID-19 diagnosis: Detection of viral proteins and host’s biological processes altered by the infection. Data in Brief. 32, 106121 DOI: 10.1016/j.dib.2020.106121
  • Sogues A, Martinez M, Gaday Q, Ben Assaya M, Graña M, Voegele A, VanNieuwenhze M, England P, Haouz A, Chenal A, Trépout S, Duran R, Wehenkel AM, Alzari PM. (2020) Essential dynamic interdependence of FtsZ and SepF for Z-ring and septum formation in Corynebacterium glutamicum Nature Communications 11, 1641 doi:10.1038/s41467-020-15490-8.
  • Rodríguez Mallón A, Javier González L, Encinosa Guzmán PE, Bechara GH, Sanches GS, Pousa S, Cabrera G, Cabrales A, Garay H, Mejías R, López Álvarez JR, Bello Soto Y, Almeida F, Guirola O, Rodríguez Fernández R, Fuentes Castillo A, Méndez L, Jiménez S, Licea-Navarro A, Portela M, Durán R, Estrada MP (2020) Functional and Mass Spectrometric Evaluation of an Anti-Tick Antigen Based on the P0 Peptide Conjugated to Bm86 Protein. Pathogens 25;9(6):513. doi:10.3390/pathogens9060513.
  • Bei J, Bigi MM, Lima A, Zhang Q, Blanco FC, Lopez B, Yu T, Wang Z, Dai Z,Chen Z, Cataldi AA, Sasiani MC, Ritacco V, de la Barrera S, Soria MA, Durán R, Bigi F. (2020) A Phenotypic characterization of two isolates of a multidrug-resistant outbreak strain of Mycobacterium tuberculosis with opposite epidemiological fitness. Biomed Res Int. 8; 2020:4741237. 6doi: 10.1155/2020/4741237.
  • Forrellad MA, Blanco FC, Marrero Diaz De Villegas R, Vázquez CL, Yaneff A, García EA, Gutierrez M, Durán R, Villarino A, Bigi F. (2020). Rv2577 of Mycobacterium tuberculosis is a virulence factor with dual phosphatase and phosphodiesterase functions. Front. Microbiol. 11:570794. doi: 10.3389/fmicb.2020.570794.
  • Carreño M, Bresque M, Machado M, Santos L, Durán R, Vitturi DA Escande C, Denicola A (2020). Nitro-fatty acids as activators of hSIRT6 deacetylase activity. Journal of Biological Chemistry 295, 18355 doi:10.1074/jbc.RA120.014883.
  • Colman L, Caggiani M, Leyva A, Bresque M, Liechocki S, Maya-Monteiro CM, Mazal D, Batthyany C, Calliari A, Contreras P, Escande C. (2020) The protein Deleted in Breast Cancer-1 (DBC1) regulates vascular response and formation of aortic dissection during Angiotensin II infusion. Sci Rep. 10, 6772 DOI: 10.1038/s41598-020-63841-8.
  • Mosquillo MF, Smircich P, Lima A, Gehrke SA, Scalese G, Machado I, Gambino D, Garat B, Pérez-Díaz L. (2020) High Throughput Approaches to Unravel the Mechanism of Action of a New Vanadium-Based Compound against Trypanosoma cruzi. Bioinorg Chem Appl. 11:1634270. doi: 10.1155/2020/1634270.
  • Mosquillo MF, Smircich P, Ciganda M, Lima A, Gambino D, Garat B, Pérez-Díaz L. (2020) Comparative high-throughput analysis of the Trypanosoma cruzi response to organometallic compounds. Metallomics. 27;12(5):813-828. doi: 10.1039/d0mt00030b.
  • Tucci P, Portela M, Rivas Chetto C, González-Sapienza G, Marín M.(2020). Integrative proteomic and glycoproteomic profiling of Mycobacterium tuberculosis culture filtrate. Plos One 15(3):e0221837. doi: 10.1371/journal.pone.0221837. eCollection 2020
2019
  • Gil M, Lima A, Rivera B, Rossello J, Urdániz E, Cascioferro A, Carrión F, Wehenkel A, Bellinzoni M, Batthyány C, Pritsch O, Denicola A, Alvarez MN, Carvalho PC, Lisa MN, Brosch R, Piuri M, Alzari PM, Durán R. (2019) New substrates and interactors of the mycobacterial Serine/Threonine protein kinase PknG identified by a tailored interactomic approach. (2019) J Proteomics. 192, 321-333. doi: 10.1016/j.jprot.2018.09.013.
  • Bellinzoni M, Wehenkel AM, Durán R, Alzari PM. (2019) Novel mechanistic insights into physiological signaling pathways mediated by mycobacterial Ser/Thr protein kinases. Microbes Infect. 21(5-6):222-229. doi: 10.1016/j.micinf.2019.06.015. Review.
  • Santos L, Colman L, Contreras P, Chini CC, Carlomagno A, Leyva A, Bresque M, Marmisolle I, Quijano C, Durán R, Irigoín F, Prieto-Echagüe V, Vendelbo MH, Sotelo-Silveira JR, Chini EN, Badano JL, Calliari AJ, Escande C. (2019) A novel form of Deleted in breast cancer 1 (DBC1) lacking the N-terminal domain does not bind SIRT1 and is dynamically regulated in vivo. Sci Rep. 9, 14381 DOI: 10.1038/s41598-019-50789-7.
  • Miles S, Portela M, Cyrklaff M, Ancarola ME, Frischknecht F, Durán R, Dematteis S, Mourglia‐Ettlin G (2019) Combining proteomics and bioinformatics to explore novel tegumental antigens as vaccine candidates against Echinococcus granulosus infection. J Cell Biochem. 120(9),15320-15336. doi: 10.1002/jcb.28799.
  • Spinelli R, Sanchis I, Aimaretti FM, Attademo AM, Portela M, Humpola MV, Tonarelli GG, Siano AS. (2019) Natural Multi-Target Inhibitors of Cholinesterases and Monoamine Oxidase Enzymes with Antioxidant Potential from Skin Extracts of Hypsiboas cordobae and Pseudis minuta (Anura: Hylidae). ChemBiodivers 16(1):e1800472. doi:10.1002/cbdv.201800472.
  • Chavarría C, Trostchansky A, Durán R, Rubbo H, Souza JM. (2019) Nitroalkylation of α-Synuclein by Nitro-Oleic Acid: Implications for Parkinson’s Disease. AdvExp Med Biol. 2019; 1127:169-179. doi: 10.1007/978-3-030-11488-6_11. Review.
  • Ramos-Artuso F, Galatro A, Lima A, Batthyány C, Simontacchi M. (2019) Early events following phosphorus restriction involve changes in proteome and affects nitric oxide metabolism in soybean leaves. Environ Exp Bot. 161:203-2017. https://doi.org/10.1016/j.envexpbot.2019.01.002.
2018
  • 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
2017
  • 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.
2013
  • 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).
2008
  • 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).
2005
  • 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.