The long slender bloodstream form Trypanosoma brucei maintains its essential mitochondrial membrane potential (ΔΨm) through the proton-pumping activity of the FoF1-ATP synthase operating in the reverse mode. The ATP that drives this hydrolytic reaction has long been thought to be generated by glycolysis and imported from the cytosol via an ATP/ADP carrier (AAC). Indeed, we demonstrate that AAC is the only carrier that can import ATP into the mitochondrial matrix to power the hydrolytic activity of the FoF1-ATP synthase. However, contrary to expectations, the deletion of AAC has no effect on parasite growth, virulence or levels of ΔΨm. This suggests that ATP is produced by substrate-level phosphorylation pathways in the mitochondrion. Therefore, we knocked out the succinyl-CoA synthetase (SCS) gene, a key mitochondrial enzyme that produces ATP through substrate-level phosphorylation in this parasite. Its absence resulted in changes to the metabolic landscape of the parasite, lowered virulence, and reduced mitochondrial ATP content. Strikingly, these SCS mutant parasites become more dependent on AAC as demonstrated by a 25-fold increase in their sensitivity to the AAC inhibitor, carboxyatractyloside. Since the parasites were able to adapt to the loss of SCS in culture, we also analyzed the more immediate phenotypes that manifest when SCS expression is rapidly suppressed by RNAi. Importantly, when performed under nutrient-limited conditions mimicking various host environments, SCS depletion strongly affected parasite growth and levels of ΔΨm. In totality, the data establish that the long slender bloodstream form mitochondrion is capable of generating ATP via substrate-level phosphorylation pathways.

Faculty of Science University of South Bohemia Ceske Budejovice Czech republic

Institute of Entomology Biology Centre CAS Ceske Budejovice Czech republic

Institute of Molecular Biology Mainz Germany

Institute of Molecular Virology and Cell Biology Friedrich Loeffler Institute Greifswald Germany

Institute of Parasitology Biology Centre CAS Ceske Budejovice Czech republic

Univ Bordeaux CNRS Centre de Résonance Magnétique des Systèmes Biologiques Bordeaux France

Univ Bordeaux CNRS Laboratoire de Microbiologie Fondamentale et Pathogénicité Université de Bordeaux Bordeaux France

Zobrazit více v PubMed

Zikova A. Mitochondrial adaptations throughout the Trypanosoma brucei life cycle. J Eukaryot Microbiol. 2022:e12911. Epub 2022/03/25. doi: 10.1111/jeu.12911 . PubMed DOI

Silva Pereira S, Trindade S, De Niz M, Figueiredo LM. Correction to ’Tissue tropism in parasitic diseases’. Open biology. 2019;9(6):190124. Epub 2019/06/27. doi: 10.1098/rsob.190124 . PubMed DOI PMC

Mantilla BS, Marchese L, Casas-Sanchez A, Dyer NA, Ejeh N, Biran M, et al.. Proline Metabolism is Essential for Trypanosoma brucei brucei Survival in the Tsetse Vector. PLoS Pathog. 2017;13(1):e1006158. Epub 2017/01/24. doi: 10.1371/journal.ppat.1006158 . PubMed DOI PMC

Millerioux Y, Ebikeme C, Biran M, Morand P, Bouyssou G, Vincent IM, et al.. The threonine degradation pathway of the Trypanosoma brucei procyclic form: the main carbon source for lipid biosynthesis is under metabolic control. Mol Microbiol. 2013;90(1):114–29. Epub 2013/08/01. doi: 10.1111/mmi.12351 . PubMed DOI PMC

Villafraz O, Biran M, Pineda E, Plazolles N, Cahoreau E, Souza ROO, et al.. Procyclic trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in the presence of physiological amounts of proline. Plos Pathogens. 2021;17(3). ARTN e1009204 doi: 10.1371/journal.ppat.1009204 PubMed DOI PMC

Bochud-Allemann N, Schneider A. Mitochondrial substrate level phosphorylation is essential for growth of procyclic Trypanosoma brucei. J Biol Chem. 2002;277(36):32849–54. Epub 2002/07/04. [pii] doi: 10.1074/jbc.M205776200 . PubMed DOI

Millerioux Y, Morand P, Biran M, Mazet M, Moreau P, Wargnies M, et al.. ATP synthesis-coupled and -uncoupled acetate production from acetyl-CoA by mitochondrial acetate:succinate CoA-transferase and acetyl-CoA thioesterase in Trypanosoma. J Biol Chem. 2012;287(21):17186–97. Epub 2012/04/05. doi: 10.1074/jbc.M112.355404 . PubMed DOI PMC

Zikova A, Schnaufer A, Dalley RA, Panigrahi AK, Stuart KD. The F(0)F(1)-ATP synthase complex contains novel subunits and is essential for procyclic Trypanosoma brucei. PLoS Pathog. 2009;5(5):e1000436. Epub 2009/05/14. doi: 10.1371/journal.ppat.1000436 . PubMed DOI PMC

Dewar CE, Casas-Sanchez A, Dieme C, Crouzols A, Haines LR, Acosta-Serrano A, et al.. Oxidative Phosphorylation Is Required for Powering Motility and Development of the Sleeping Sickness Parasite Trypanosoma brucei in the Tsetse Fly Vector. mBio. 2022:e0235721. Epub 2022/01/12. doi: 10.1128/mbio.02357-21 . PubMed DOI PMC

Michels PAM, Villafraz O, Pineda E, Alencar MB, Caceres AJ, Silber AM, et al.. Carbohydrate metabolism in trypanosomatids: New insights revealing novel complexity, diversity and species-unique features. Exp Parasitol. 2021;224:108102. Epub 2021/03/30. doi: 10.1016/j.exppara.2021.108102 . PubMed DOI

Zikova A, Verner Z, Nenarokova A, Michels PAM, Lukes J. A paradigm shift: The mitoproteomes of procyclic and bloodstream Trypanosoma brucei are comparably complex. PLoS Pathog. 2017;13(12):e1006679. Epub 2017/12/22. doi: 10.1371/journal.ppat.1006679 . PubMed DOI PMC

Surve S, Heestand M, Panicucci B, Schnaufer A, Parsons M. Enigmatic presence of mitochondrial complex I in Trypanosoma brucei bloodstream forms. Eukaryot Cell. 2012;11(2):183–93. Epub 2011/12/14. doi: 10.1128/EC.05282-11 . PubMed DOI PMC

Chaudhuri M, Ott RD, Hill GC. Trypanosome alternative oxidase: from molecule to function. Trends Parasitol. 2006;22(10):484–91. Epub 2006/08/22. S1471-4922(06)00212-1 [pii] doi: 10.1016/j.pt.2006.08.007 . PubMed DOI

Gualdron-Lopez M, Brennand A, Hannaert V, Quinones W, Caceres AJ, Bringaud F, et al.. When, how and why glycolysis became compartmentalised in the Kinetoplastea. A new look at an ancient organelle. Int J Parasitol. 2012;42(1):1–20. Epub 2011/12/07. doi: 10.1016/j.ijpara.2011.10.007 . PubMed DOI

Nolan DP, Voorheis HP. The mitochondrion in bloodstream forms of Trypanosoma brucei is energized by the electrogenic pumping of protons catalysed by the F1F0-ATPase. Eur J Biochem. 1992;209(1):207–16. Epub 1992/10/01. doi: 10.1111/j.1432-1033.1992.tb17278.x . PubMed DOI

Schnaufer A, Clark-Walker GD, Steinberg AG, Stuart K. The F1-ATP synthase complex in bloodstream stage trypanosomes has an unusual and essential function. EMBO J. 2005;24(23):4029–40. Epub 2005/11/05. 7600862 [pii] doi: 10.1038/sj.emboj.7600862 . PubMed DOI PMC

Chinopoulos C, Adam-Vizi V. Mitochondria as ATP consumers in cellular pathology. Biochim Biophys Acta. 2010;1802(1):221–7. Epub 2009/09/01. doi: 10.1016/j.bbadis.2009.08.008 . PubMed DOI

Chinopoulos C. Mitochondrial consumption of cytosolic ATP: Not so fast. Febs Letters. 2011;585(9):1255–9. doi: 10.1016/j.febslet.2011.04.004 PubMed DOI

Chinopoulos C. The "B space" of mitochondrial phosphorylation. J Neurosci Res. 2011;89(12):1897–904. Epub 2011/05/05. doi: 10.1002/jnr.22659 . PubMed DOI

St-Pierre J, Brand MD, Boutilier RG. Mitochondria as ATP consumers: cellular treason in anoxia. Proc Natl Acad Sci U S A. 2000;97(15):8670–4. Epub 2000/07/13. [pii] doi: 10.1073/pnas.140093597 . PubMed DOI PMC

Chen WW, Birsoy K, Mihaylova MM, Snitkin H, Stasinski I, Yucel B, et al.. Inhibition of ATPIF1 ameliorates severe mitochondrial respiratory chain dysfunction in mammalian cells. Cell reports. 2014;7(1):27–34. Epub 2014/04/02. doi: 10.1016/j.celrep.2014.02.046 . PubMed DOI PMC

Hierro-Yap C, Subrtova K, Gahura O, Panicucci B, Dewar C, Chinopoulos C, et al.. Bioenergetic consequences of FoF1-ATP synthase/ATPase deficiency in two life cycle stages of Trypanosoma brucei. J Biol Chem. 2021;296:100357. Epub 2021/02/05. doi: 10.1016/j.jbc.2021.100357 . PubMed DOI PMC

Subrtova K, Panicucci B, Zikova A. ATPaseTb2, a Unique Membrane-bound FoF1-ATPase Component, Is Essential in Bloodstream and Dyskinetoplastic Trypanosomes. PLoS Pathog. 2015;11(2):e1004660. Epub 2015/02/26. doi: 10.1371/journal.ppat.1004660 . PubMed DOI PMC

Panicucci B, Gahura O, Zikova A. Trypanosoma brucei TbIF1 inhibits the essential F1-ATPase in the infectious form of the parasite. PLoS Negl Trop Dis. 2017;11(4):e0005552. Epub 2017/04/18. doi: 10.1371/journal.pntd.0005552 . PubMed DOI PMC

Pena-Diaz P, Pelosi L, Ebikeme C, Colasante C, Gao F, Bringaud F, et al.. Functional characterization of TbMCP5, a conserved and essential ADP/ATP carrier present in the mitochondrion of the human pathogen Trypanosoma brucei. J Biol Chem. 2012;287(50):41861–74. Epub 2012/10/18. doi: 10.1074/jbc.M112.404699 . PubMed DOI PMC

Gnipova A, Subrtova K, Panicucci B, Horvath A, Lukes J, Zikova A. The ADP/ATP carrier and its relationship to OXPHOS in an ancestral protist, Trypanosoma brucei. Eukaryot Cell. 2015. Epub 2015/01/27. PubMed PMC

Vickerman K. Polymorphism and mitochondrial activity in sleeping sickness trypanosomes. Nature. 1965;208(5012):762–6. Epub 1965/11/20. doi: 10.1038/208762a0 . PubMed DOI

Prochazkova M, Panicucci B, Zikova A. Cultured bloodstream Trypanosoma brucei adapt to life without mitochondrial translation release factor 1. Scientific reports. 2018;8(1):5135. Epub 2018/03/25. doi: 10.1038/s41598-018-23472-6 . PubMed DOI PMC

Dean S, Gould MK, Dewar CE, Schnaufer AC. Single point mutations in ATP synthase compensate for mitochondrial genome loss in trypanosomes. Proc Natl Acad Sci U S A. 2013;110(36):14741–6. Epub 2013/08/21. doi: 10.1073/pnas.1305404110 . PubMed DOI PMC

Dejung M, Subota I, Bucerius F, Dindar G, Freiwald A, Engstler M, et al.. Quantitative Proteomics Uncovers Novel Factors Involved in Developmental Differentiation of Trypanosoma brucei. PLoS Pathog. 2016;12(2):e1005439. Epub 2016/02/26. doi: 10.1371/journal.ppat.1005439 . PubMed DOI PMC

Johnston K, Kim DH, Kerkhoven EJ, Burchmore R, Barrett MP, Achcar F. Mapping the metabolism of five amino acids in bloodstream form Trypanosoma brucei using U-C-13-labelled substrates and LC-MS. Bioscience Rep. 2019;39. Artn Bsr20181601 doi: 10.1042/Bsr20181601 PubMed DOI PMC

Creek DJ, Mazet M, Achcar F, Anderson J, Kim DH, Kamour R, et al.. Probing the metabolic network in bloodstream-form Trypanosoma brucei using untargeted metabolomics with stable isotope labelled glucose. PLoS Pathog. 2015;11(3):e1004689. Epub 2015/03/17. doi: 10.1371/journal.ppat.1004689 . PubMed DOI PMC

Mazet M, Morand P, Biran M, Bouyssou G, Courtois P, Daulouede S, et al.. Revisiting the central metabolism of the bloodstream forms of Trypanosoma brucei: production of acetate in the mitochondrion is essential for parasite viability. PLoS Negl Trop Dis. 2013;7(12):e2587. Epub 2013/12/25. doi: 10.1371/journal.pntd.0002587 . PubMed DOI PMC

Van Hellemond JJ, Opperdoes FR, Tielens AGM. Trypanosomatidae produce acetate via a mitochondrial acetate: succinate CoA transferase. P Natl Acad Sci USA. 1998;95(6):3036–41. doi: 10.1073/pnas.95.6.3036 PubMed DOI PMC

Bienen EJ, Maturi RK, Pollakis G, Clarkson AB Jr. Non-cytochrome mediated mitochondrial ATP production in bloodstream form Trypanosoma brucei brucei. Eur J Biochem. 1993;216(1):75–80. Epub 1993/08/15. doi: 10.1111/j.1432-1033.1993.tb18118.x . PubMed DOI

Franco JR, Cecchi G, Paone M, Diarra A, Grout L, Kadima Ebeja A, et al.. The elimination of human African trypanosomiasis: Achievements in relation to WHO road map targets for 2020. PLoS Negl Trop Dis. 2022;16(1):e0010047. Epub 2022/01/19. doi: 10.1371/journal.pntd.0010047 . PubMed DOI PMC

Alkhaldi AA, Martinek J, Panicucci B, Dardonville C, Zikova A, de Koning HP. Trypanocidal action of bisphosphonium salts through a mitochondrial target in bloodstream form Trypanosoma brucei. International journal for parasitology Drugs and drug resistance. 2016;6(1):23–34. Epub 2016/04/08. doi: 10.1016/j.ijpddr.2015.12.002 . PubMed DOI PMC

Lanteri CA, Tidwell RR, Meshnick SR. The mitochondrion is a site of trypanocidal action of the aromatic diamidine DB75 in bloodstream forms of Trypanosoma brucei. Antimicrob Agents Chemother. 2008;52(3):875–82. Epub 2007/12/19. AAC.00642-07 [pii] doi: 10.1128/AAC.00642-07 . PubMed DOI PMC

Eze AA, Gould MK, Munday JC, Tagoe DN, Stelmanis V, Schnaufer A, et al.. Reduced Mitochondrial Membrane Potential Is a Late Adaptation of Trypanosoma brucei brucei to Isometamidium Preceded by Mutations in the gamma Subunit of the F1Fo-ATPase. PLoS Negl Trop Dis. 2016;10(8):e0004791. Epub 2016/08/16. doi: 10.1371/journal.pntd.0004791 . PubMed DOI PMC

Carruthers LV, Munday JC, Ebiloma GU, Steketee P, Jayaraman S, Campagnaro GD, et al.. Diminazene resistance in Trypanosoma congolense is not caused by reduced transport capacity but associated with reduced mitochondrial membrane potential. Mol Microbiol. 2021. Epub 2021/05/02. doi: 10.1111/mmi.14733 . PubMed DOI

Wilkes JM, Mulugeta W, Wells C, Peregrine AS. Modulation of mitochondrial electrical potential: a candidate mechanism for drug resistance in African trypanosomes. Biochem J. 1997;326 (Pt 3)(Pt 3):755–61. Epub 1997/10/23. doi: 10.1042/bj3260755 . PubMed DOI PMC

Creek DJ, Nijagal B, Kim DH, Rojas F, Matthews KR, Barrett MP. Metabolomics guides rational development of a simplified cell culture medium for drug screening against Trypanosoma brucei. Antimicrob Agents Chemother. 2013;57(6):2768–79. Epub 2013/04/11. doi: 10.1128/AAC.00044-13 . PubMed DOI PMC

Figueira TR, Melo DR, Vercesi AE, Castilho RF. Safranine as a fluorescent probe for the evaluation of mitochondrial membrane potential in isolated organelles and permeabilized cells. Methods Mol Biol. 2012;810:103–17. Epub 2011/11/08. doi: 10.1007/978-1-61779-382-0_7 . PubMed DOI

Mach J, Poliak P, Matuskova A, Zarsky V, Janata J, Lukes J, et al.. An Advanced System of the Mitochondrial Processing Peptidase and Core Protein Family in Trypanosoma brucei and Multiple Origins of the Core I Subunit in Eukaryotes. Genome Biol Evol. 2013;5(5):860–75. Epub 2013/04/09. doi: 10.1093/gbe/evt056 . PubMed DOI PMC

Alexander PB, Wang J, McKnight SL. Targeted killing of a mammalian cell based upon its specialized metabolic state. Proc Natl Acad Sci U S A. 2011;108(38):15828–33. Epub 2011/09/08. doi: 10.1073/pnas.1111312108 . PubMed DOI PMC

Elkalaf M, Tuma P, Weiszenstein M, Polak J, Trnka J. Mitochondrial Probe Methyltriphenylphosphonium (TPMP) Inhibits the Krebs Cycle Enzyme 2-Oxoglutarate Dehydrogenase. PLoS One. 2016;11(8):e0161413. Epub 2016/08/19. doi: 10.1371/journal.pone.0161413 . PubMed DOI PMC

Pineda E, Thonnus M, Mazet M, Mourier A, Cahoreau E, Kulyk H, et al.. Glycerol supports growth of the Trypanosoma brucei bloodstream forms in the absence of glucose: Analysis of metabolic adaptations on glycerol-rich conditions. PLoS Pathog. 2018;14(11):e1007412. Epub 2018/11/02. doi: 10.1371/journal.ppat.1007412 . PubMed DOI PMC

Kovarova J, Nagar R, Faria J, Ferguson MAJ, Barrett MP, Horn D. Gluconeogenesis using glycerol as a substrate in bloodstream-form Trypanosoma brucei. PLoS Pathog. 2018;14(12):e1007475. Epub 2018/12/28. doi: 10.1371/journal.ppat.1007475 . PubMed DOI PMC

Trindade S, Rijo-Ferreira F, Carvalho T, Pinto-Neves D, Guegan F, Aresta-Branco F, et al.. Trypanosoma brucei Parasites Occupy and Functionally Adapt to the Adipose Tissue in Mice. Cell Host Microbe. 2016;19(6):837–48. Epub 2016/05/31. doi: 10.1016/j.chom.2016.05.002 . PubMed DOI PMC

Capewell P, Cren-Travaille C, Marchesi F, Johnston P, Clucas C, Benson RA, et al.. The skin is a significant but overlooked anatomical reservoir for vector-borne African trypanosomes. eLife. 2016;5. Epub 2016/09/23. doi: 10.7554/eLife.17716 . PubMed DOI PMC

Mochizuki K, Inaoka DK, Mazet M, Shiba T, Fukuda K, Kurasawa H, et al.. The ASCT/SCS cycle fuels mitochondrial ATP and acetate production in Trypanosoma brucei. Bba-Bioenergetics. 2020;1861(11). ARTN 148283 doi: 10.1016/j.bbabio.2020.148283 PubMed DOI PMC

Nascimento JF, Souza ROO, Alencar MB, Marsiccobetre S, Murillo AM, Damasceno FS, et al.. How much (ATP) does it cost to build a trypanosome? A theoretical study on the quantity of ATP needed to maintain and duplicate a bloodstream-form Trypanosoma brucei cell. PLoS Pathog. 2023;19(7):e1011522. Epub 2023/07/27. doi: 10.1371/journal.ppat.1011522 . PubMed DOI PMC

Riviere L, Moreau P, Allmann S, Hahn M, Biran M, Plazolles N, et al.. Acetate produced in the mitochondrion is the essential precursor for lipid biosynthesis in procyclic trypanosomes. Proc Natl Acad Sci U S A. 2009;106(31):12694–9. Epub 2009/07/25. doi: 10.1073/pnas.0903355106 . PubMed DOI PMC

Millerioux Y, Mazet M, Bouyssou G, Allmann S, Kiema TR, Bertiaux E, et al.. De novo biosynthesis of sterols and fatty acids in the Trypanosoma brucei procyclic form: Carbon source preferences and metabolic flux redistributions. PLoS Pathog. 2018;14(5):e1007116. Epub 2018/05/31. doi: 10.1371/journal.ppat.1007116 . PubMed DOI PMC

Sykes SE, Hajduk SL. Dual functions of alpha-ketoglutarate dehydrogenase E2 in the Krebs cycle and mitochondrial DNA inheritance in Trypanosoma brucei. Eukaryot Cell. 2013;12(1):78–90. Epub 2012/11/06. doi: 10.1128/EC.00269-12 . PubMed DOI PMC

Spitznagel D, Ebikeme C, Biran M, Nic a’ Bhaird N, Bringaud F, Henehan GT, et al.. Alanine aminotransferase of Trypanosoma brucei—a key role in proline metabolism in procyclic life forms. FEBS J. 2009;276(23):7187–99. Epub 2009/11/10. doi: 10.1111/j.1742-4658.2009.07432.x . PubMed DOI

Steiger RF, Opperdoes FR, Bontemps J. Subcellular fractionation of Trypanosoma brucei bloodstream forms with special reference to hydrolases. Eur J Biochem. 1980;105(1):163–75. Epub 1980/03/01. doi: 10.1111/j.1432-1033.1980.tb04486.x . PubMed DOI

Billington K, Halliday C, Madden R, Dyer P, Barker AR, Moreira-Leite FF, et al.. Genome-wide subcellular protein map for the flagellate parasite Trypanosoma brucei. Nature microbiology. 2023;8(3):533–47. Epub 2023/02/23. doi: 10.1038/s41564-022-01295-6 . PubMed DOI PMC

Pyrih J, Hammond M, Alves A, Dean S, Sunter JD, Wheeler RJ, et al.. Comprehensive sub-mitochondrial protein map of the parasitic protist Trypanosoma brucei defines critical features of organellar biology. Cell reports. 2023;42(9):113083. Epub 2023/09/05. doi: 10.1016/j.celrep.2023.113083 . PubMed DOI

Wirtz E, Leal S, Ochatt C, Cross GA. A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol Biochem Parasitol. 1999;99(1):89–101. Epub 1999/04/24. S016668519900002X [pii]. doi: 10.1016/s0166-6851(99)00002-x . PubMed DOI

Wickstead B, Ersfeld K, Gull K. Targeting of a tetracycline-inducible expression system to the transcriptionally silent minichromosomes of Trypanosoma brucei. Mol Biochem Parasitol. 2002;125(1–2):211–6. Epub 2002/12/07. S0166685102002384 [pii]. doi: 10.1016/s0166-6851(02)00238-4 . PubMed DOI

Riviere L, van Weelden SW, Glass P, Vegh P, Coustou V, Biran M, et al.. Acetyl:succinate CoA-transferase in procyclic Trypanosoma brucei. Gene identification and role in carbohydrate metabolism. J Biol Chem. 2004;279(44):45337–46. Epub 2004/08/25. M407513200 [pii] doi: 10.1074/jbc.M407513200 . PubMed DOI

Skodova-Sverakova I, Zahonova K, Juricova V, Danchenko M, Moos M, Barath P, et al.. Highly flexible metabolism of the marine euglenozoan protist Diplonema papillatum. BMC biology. 2021;19(1):251. Epub 2021/11/26. doi: 10.1186/s12915-021-01186-y . PubMed DOI PMC

Moos M, Korbelova J, Stetina T, Opekar S, Simek P, Grgac R, et al.. Cryoprotective Metabolites Are Sourced from Both External Diet and Internal Macromolecular Reserves during Metabolic Reprogramming for Freeze Tolerance in Drosophilid Fly, Chymomyza costata. Metabolites. 2022;12(2). Epub 2022/02/26. doi: 10.3390/metabo12020163 . PubMed DOI PMC

Haug K, Cochrane K, Nainala VC, Williams M, Chang J, Jayaseelan KV, et al.. MetaboLights: a resource evolving in response to the needs of its scientific community. Nucleic Acids Res. 2020;48(D1):D440–D4. Epub 2019/11/07. doi: 10.1093/nar/gkz1019 . PubMed DOI PMC

Rappsilber J, Mann M, Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nature protocols. 2007;2(8):1896–906. Epub 2007/08/19. doi: 10.1038/nprot.2007.261 . PubMed DOI

Perez-Riverol Y, Bai J, Bandla C, Garcia-Seisdedos D, Hewapathirana S, Kamatchinathan S, et al.. The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res. 2022;50(D1):D543–D52. Epub 2021/11/02. doi: 10.1093/nar/gkab1038 . PubMed DOI PMC

Phenotypic screening reveals a highly selective phthalimide-based compound with antileishmanial activity