Computational protocol: Cellular Growth and Mitochondrial Ultrastructure of Leishmania (Viannia) braziliensis Promastigotes Are Affected by the Iron Chelator 2,2-Dipyridyl

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[…] For the first dimension, 500 µg protein was diluted to a final volume of 350 µl in rehydration solution (9 M urea, 4% CHAPS, 65 mM DTT, 1.5% ampholytes pH 3–10, 0,001% bromophenol blue). This solution was applied to IEF strips (18 cm pH 3–10 nonlinear; GE Healthcare) and submitted to isoelectric focalization at Ettan IPGphor 3 (GE Healthcare) at 20°C and a maximum current of 50 µA/strip. Focusing parameters and the second dimension were set as previously described . Detection of spots and comparison of protein expression were performed using PDQuest software (Bio-Rad). The intensity of each spot, measured in parts per million (ppm), provided the basis for comparison of protein expression in parasites cultured in control medium or treated with the iron chelator. To normalize the intensity values, the pixel intensity of each spot, measured in ppm, was divided by the total intensity of all pixels present in the image. [...] Protein spots were manually excised and treated for digestion. Gel pieces were washed three times in 400 µL of 50% acetonitrile, 25 mM NH4HCO3 pH 8,0dehydrated in acetonitrile 100% and dried in a vacuum centrifuge. Gel pieces were rehydrated in 15 µl of 50 mM NH4HCO3 with 200 ng of trypsin (Promega). After 15 min, 20 µl of 50 mM a NH4HCO3 was added to keep gel pieces wet during tryptic digestion (37°C, overnight). To extract peptides, 20 µl of 0.5% (v/v) trifluoroacetic acid (TFA) in 50% (v/v) acetonitrile were added, and samples were sonicated for 30 min. The separated liquid was concentrated under a vacuum to an approximate volume of 10 µl. Tryptic peptides were purified using ZipTip C18 pipette tips following the manufacturer's instructions (Millipore), eluted with 3.0 µl of 0.1% (v/v) TFA in 50% (v/v) acetonitrile and co-crystallized with matrix (7 mg/mL alpha-cyano-4-hydroxycinnamic acid) on a stainless-steel plate using 0.5 µl of matrix and 0.5 µl of sample. Mass spectra were acquired on a 5800 Proteomics Analyzer mass spectrometer (MALDI-TOF/TOF™, Applied Biosystems) operating in delayed reflector mode with an accelerated voltage of 20 kV. MS/MS spectra corresponding to the five most intense signals were obtained automatically using the CID acquisition mode. Proteins were identified by searching in the non-redundant database of the National Center for Biotechnology Information (NCBInr) using the program Mascot MS/MS ion search (Matrix Science, Oxford, UK, www.matrixscience.com/search_form_select.html). The search parameters in the Mascot server were lack of taxonomic restrictions; permission of tryptic peptides with only one erroneous cleavage; carbamidomethylation of cysteine residues as a fixed modification and oxidation of methionine as a variable modification; 100 ppm mass tolerance for the MS mode; and 0.2 Da tolerance for its corresponding fragments in MS/MS. Proteins identified as hypothetical and therefore of unknown function were analyzed using InterProScan Sequence Search Tool (http://www.ebi.ac.uk/Tools/pfa/iprscan/) from the InterPro data library (http://www.ebi.ac.uk/Interpro/) to assign predicted functional domains . This data library combines independent databases to generate an integrated source of information about protein families, domains, sites and regions, thus enhancing annotation. The data were analyzed following the standards proposed under the Minimum Information About a Proteomic Experiment MIAPE consensus . […]

Pipeline specifications

Software tools PDQuest 2-D, Mascot Server, InterProScan
Applications MS-based untargeted proteomics, Proteome data visualization
Organisms Leishmania braziliensis
Diseases Leishmaniasis, Mitochondrial Diseases
Chemicals Iron, Propidium, 2,2'-Dipyridyl