title: Cell disruption using a different methodology for proteomics analysis of Trypanosoma cruzi strains.
abstract: We have developed a cell disruption method to produce a protein extract using Trypanosoma cruzi cells based on a straightforward hypoosmotic lysis protocol. The procedure consists of three steps: incubation of the cells in a hypoosmotic lysis buffer, sonication in a water bath, and centrifugation. The final protein extract was designated TcS12. The stages of cell disruption at different incubation times were monitored by differential interference contrast microscopy. After 30min of incubation in lysis buffer at 4°C, the T. cruzi epimastigote forms changed from slender to round-shaped parasites. Nevertheless, cell disruption took place following sonication of the sample for 30min. The efficiency of the methodology was also validated by flow cytometry, which resulted in 72% of propidium iodide (PI)-labeled cells. To estimate the protein extraction yield and the differential protein expression, the proteomics profile of four T. cruzi strains (CL-Brener, Dm28c, Y, and 4167) were analyzed by liquid chromatography tandem mass spectrometry (LCMS/MS) on a SYNAPT HDMS system using the label-free MS(E) approach. ProteinLynx Global Server (version 2.5) with Expression(E) analysis identified a total of 1153 proteins and revealed 428 differentially expressed proteins among the strains. Gene ontology analysis showed that not only cytosolic proteins but also nuclear and organellar ones were present in the extract.
authors.affiliation: Laboratório Interdisciplinar de Pesquisas Médicas, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz (FIOCRUZ), 21040-360 Manguinhos, Rio de Janeiro, RJ, Brazil.
authors.forename: Tainah
authors.initials: T
authors.lastname: Silva Galdino
authors.affiliation: Laboratório de Biologia Celular, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz (FIOCRUZ), 21040-360 Manguinhos, Rio de Janeiro, RJ, Brazil.
authors.forename: Rubem Figueiredo Sadok
authors.initials: RF
authors.lastname: Menna-Barreto
authors.affiliation: Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz (FIOCRUZ), 21040-360 Manguinhos, Rio de Janeiro, RJ, Brazil.
authors.forename: Constança
authors.initials: C
authors.lastname: Britto
authors.affiliation: Laboratório Interdisciplinar de Pesquisas Médicas, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz (FIOCRUZ), 21040-360 Manguinhos, Rio de Janeiro, RJ, Brazil; Laboratório de Parasitologia, Instituto Conmemorativo Gorgas de Estudios de la Salud (ICGES), 0816-02593, Panama.
authors.forename: Franklyn
authors.initials: F
authors.lastname: Samudio
authors.affiliation: Laboratório Interdisciplinar de Pesquisas Médicas, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz (FIOCRUZ), 21040-360 Manguinhos, Rio de Janeiro, RJ, Brazil.
authors.forename: Adeilton
authors.initials: A
authors.lastname: Brandão
authors.affiliation: Laboratório Interdisciplinar de Pesquisas Médicas, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz (FIOCRUZ), 21040-360 Manguinhos, Rio de Janeiro, RJ, Brazil. Electronic address: kalume@ioc.fiocruz.br.
authors.forename: Dário Eluan
authors.initials: DE
authors.lastname: Kalume
mesh_terms: D002851:Chromatography, High Pressure Liquid; D005434:Flow Cytometry; D008857:Microscopy, Interference; D009997:Osmotic Pressure; D011419:Propidium / Q000737:chemistry; D020543:Proteome / Q000032:analysis*; D040901:Proteomics; D015800:Protozoan Proteins / Q000302:isolation & purification / Q000378:metabolism; D013010:Sonication; D053719:Tandem Mass Spectrometry; D014349:Trypanosoma cruzi / Q000378:metabolism*
chemical_list: D020543:Proteome; D015800:Protozoan Proteins; D011419:Propidium
keywords: Flow cytometry; Hypotonic lysis buffer; Mass spectrometry; Proteomics; Trypanosoma cruzi; Water bath sonication