| Metadata |
| datasetIdentifier | PASS00393 |
| datasetType | MSMS |
| submitter | Júnior Fialho <junior-fialho@hotmail.com> |
| submitter_organization | Universidade Federal de Minas Gerais |
| lab_head_full_name | Hélida Monteiro de Andrade |
| lab_head_email | helidandrade@gmail.com |
| lab_head_organization | Laboratory of Leishmaniosis |
| lab_head_country | Brazil |
| datasetTag | BH46xBH400 |
| datasetTitle | Identification of virulence factors in Leishmania infantum strains by a proteomic approach. |
| publicReleaseDate | 2014-01-17 00:00:00 |
| finalizedDate | 2014-01-21 09:11:18 |
| summary | The study of Leishmania virulence is essential for understanding how the contact between the pathogen and host cells can lead to pathogenesis. We characterized the virulence in two L. infantum strains using murine macrophages and infected hamsters. Next, we used 2-D DIGE and mass spectrometry to identify the differentially expressed proteins. A total of 63 spots were identified corresponding to 36 proteins; 20 were up-regulated, and 16 had been previously associated with virulence Leishmania. |
| contributors | Simone F. Pires;
Luiz C. Fialho;
Soraia O. Silva;
Maria N. Melo;
Carolina Carvalho;
Wagner L. Tafuri;
Oscar B. Romero;
Hélida M. Andrade. |
| publication | Pires, SF, and Fialho, LC; Identification of virulence factors in Leishmania infantum strains by a proteomic approach, Journal of Proteome Research, submitted. |
| growth | Promastigotes of L. infantum MHOM/BR/1972/BH6 (BH46) and MCAN/BR/2000/BH400 (BH400) strains were isolated form hamster spleens that were previously infected and grown at 25°C in α-Mem medium (Cultilab) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Cultilab), 0.4 g/L NaHCO3, 4 g/L HEPES, 200 U/mL penicillin (Cultilab), and 100 µg/mL streptomycin (Cultilab), pH 7.4. Both strains were cultivated under identical conditions (exponential growth phase, temperature, parasite concentration and medium) until the 5th passage. |
| treatment | |
| extraction | For each pair of samples (MHOM/BR/1972/BH6 and MCAN/BR/2000/BH400 strains), parasites from three independent cultures were obtained and washed three times in RPMI medium by centrifugation at 8000×g for 20 min at 4°C and maintained at -80 °C for the protein extract preparation. The parasites were suspended in 500 μL Lysis buffer (8 M urea, 2 M thiourea, 4% CHAPS, 65 mM dithiothreitol (DTT), 40 mM Tris base and a protease inhibitor mix (GE Healthcare, USA)) per 109 parasite forms. After 1 hour shaking at room temperature, the protein extract was centrifuged at 20 000×g for 1 h, and the supernatant was kept at -80 °C until use. Protein concentration was determined using the 2D Quant Kit (GE Healthcare, USA) according to the manufacturer’s instructions. The protein aliquots were stored at −80 °C until analysis. |
| separation | DIGE - Two-Dimensional Gel Electrophoresis (2-DE) - To identify differentially expressed proteins between BH46 and BH400 strains, 50 µg sample was labeled with 400 pmol N-hydroxysuccinimidyl-ester derivates of cyanine dyes Cy2, Cy3 and Cy5 (GE Healthcare, USA) following the manufacturer’s protocol. The reaction was quenched with 1 mL 10 mM lysine for 10 min on ice in the dark. A mixture of protein extracts from BH46 and BH400 was labeled with Cy2 as an internal standard. Protein extract from one strain was labeled with Cy3, and the other with Cy5. Three biological replicate experiments were performed and a dye-swap was used for both strains.
Differentially labeled extracts were pooled, reduced with 2% DTT, complemented with 2% ampholytes (pH 4–7), adjusted to a final volume of 350 µL with sample buffer (7 M urea, 2 M thiourea and 4% CHAPS), and incubated for 20 min on ice in the dark. Samples were then applied to IPG strips (18 cm, pH 4-7 NL; GE Healthcare, USA) for passive rehydration overnight at room temperature. Rehydrated IPG strips were subjected to IEF for 40,000 Vh on an Ettan IPGphor system (GE Healthcare, USA) at 20ºC and a maximum current of 50μA/strip. Focused IPG strips were equilibrated for 15 minutes in equilibration solution (50 mM Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue and 125 mM DTT) and then alkylated for a further 15 minutes in an equilibration solution containing 13.5 mM iodoacetamide instead of DTT.
Strips were transferred onto a 12% SDS-PAGE gel within low-fluorescence glass plates (GE Healthcare, USA), and second dimensional focusing was performed at 10 ºC using 20 mA/gel for 1 h, followed by 50 mA/gel, with an Ettan DALT 6 unit (GE Healthcare, USA) until the dye front reached the bottom of the gel. Electrophoresis was performed in Tris/glycine/SDS buffer and in the dark. |
| digestion | Spots with differential expression were manually excised, trypsinized and desalted using Zip-Tips (C18 resin; P10, Millipore Corporation, Bedford, MA) according to Costa et al., 2011. Approximately 0.5 μL sample solution was mixed with 0.25 μL saturated matrix solution [10 mg/mL α-cyano-4-hydroxycinnamic acid (Aldrich, Milwaukee, WI) in 50% acetonitrile/0.1% trifluoroacetic acid]. Samples were spotted on MTP AnchorChipTM 600/384 (Bruker Daltonics) targets and allowed to dry at room temperature. Raw data for the identification of proteins were obtained on a MALDI-TOF/TOF AutoFlex III™ (Bruker Daltonics, Billerica, USA) instrument in the positive/reflector mode controlled by FlexControl™ software. Instrument calibration was achieved using peptide calibration standard II (Bruker Daltonics) as a reference. Trypsin and keratin contamination peaks were excluded from the peak lists that were used in the database searching. Each spectrum was produced by accumulating data from 200 consecutive laser shots. |
| acquisition | Uninterpreted tandem mass spectra were searched against the nonredundant protein sequence database from the National Center for Biotechnology Information (NCBI) using the MASCOT® software (version 2.1) MS/MS ion search tool (http:// www.matrixscience.com). |
| informatics | |
| instruments | MALDI-ToF/ToF |
| species | Leishmania infantum |
| massModifications | ubiquitination, phosphorylation |
