Mascot program

Spectra were calibrated using a lock mass signal (m/z 1221.99064) prior to compound detection and peak list creation. The peak list files obtained from individual gel sections were combined and then submitted for database searching to a locally-running copy of the Mascot program (Matrix Science Ltd., version 2.3.02), through the ProteinScape interface (Bruker Daltonics., version 2.1). The database searched was IPI.mouse (v3.87 27/11/2011). Search criteria included: enzyme, trypsin; missed cleavages, 1; fixed modifications, carbamidomethyl (C); variable modifications, acetyl (N-terminal) and oxidation (M); peptide tolerance, 10 ppm; MS/MS tolerance, 0.1 Da. The search included an automatic decoy database search and the false discovery rate for identity was <2%. The significance threshold was p < 0.05 and the peptide ion score cut-off was 20.

GUTK or DMSO treated HepG2 cells were harvested and the protein concentration was determined using BioRad Dc protein Assay. A 2-DE was performed as described previously [33 (link)]. Briefly, the samples containing 150 μg of protein were diluted in a rehydration buffer, loaded onto IPG strips and rehydrated with an IPGphor II apparatus (Amersham). The isoelectric focusing was carried out in a stepwise voltage increasing manner. The gels were visualized by silver staining and the raw images were captured by using a GS-800 scanner and QuantityOne program, and subsequently analyzed by the PDQuest program (version 8.0, BioRad). The differentially expressed proteins spots were manually excised. After enzymatic digestion, the peptide samples were analyzed using a 4700 Proteomics Analyzer (TOF/TOF^™) (Applied Biosystems). A peptide mass mapping was performed using a MASCOT program (Matrix Science, London) against Swiss-Prot database with a GPS explorer software (Applied Biosystems).

Peak lists in .raw format were imported into Progenesis QI and LC-MS runs aligned to the common sample pool. Precursor ion intensities were normalized against total intensity for each acquisition. A combined peak list was exported in .mgf format for database searching against L. mexicana sequences appended with common proteomic contaminants (8365 sequences). MascotDaemon (version 2.5.1, Matrix Science) was used to submit the search to a locally-run copy of the Mascot program (Matrix Science Ltd., version 2.5.1). Search criteria specified: Enzyme, trypsin; Fixed modifications, Carbamidomethyl (C); Variable modifications, Oxidation (M); Peptide tolerance, 5 ppm; MS/MS tolerance, 0.5 Da; Instrument, ESI-TRAP. Search results were filtered to require a minimum expect score of 0.05. The Mascot .XML result file was imported into Progenesis QI and peptide identifications associated with precursor peak areas. Relative protein quantification was derived from unique peptide precursor ion intensities. Accepted quantifications were required to contain a minimum of two unique peptides. Statistical testing was performed in Progenesis QI and ANOVA-derived p values were converted to multiple test-corrected q-values using the Hochberg and Benjamini approach. Final quantification results were stripped of non-Leishmania spp. identifications for brevity.

Peptide mass fingerprinting (PMF) involved mass determination of tryptic fragments using MS in combination with mass database matching for peptide identifications. MS was performed using the Applied Biosystems5800 Proteomics Analyzer MALDI‐TOF/TOF (Applied Biosystems, Framingham, MA). Tryptic peptide mixtures (0.5 ml) were spotted on a 192‐well target plate and crystallized with 0.5 ml of α‐Cyano‐4‐hydroxycinnamic acid (CHCA) matrix solution (5 mg/mL). MS data were automatically acquired with a trypsin auto digest exclusion list and the 10 most intense ions selected for MS/MS. The collision gas was nitrogen air and the energy was 1 kV. Interpretation was carried out using the GPS Explorer software and database searching was done using the MASCOT program (Matrix Science, London, UK). Combined MS‐MS/MS search were conducted with the following settings: NCBI database, all entries, peptide tolerance at 200 ppm, MS/MS tolerance at 0.5 Da, carbamidomethylation of cysteine (fixed modification), and methionine oxidation (variable modifications).

The spots were excised manually from the gel and followed by trypsin digestion using a trypsin digestion kit (Pierce, USA). The trypsin digested samples were lyophilised and 5 µL of 0.1% TFA and 50% acetonitrile solution was used to dissolve the samples. A volume of 1 µL of the sample was taken for the identification of respective peptide using the MALDI-TOF-MS/MS analyser (Bruker Daltonics, Germany) and the peptide identification was performed using the MALDI-TOF-MS/MS analyser (Bruker Daltonics, Germany). Spectra of the peptides were collected using Flex Control software and Flex Analysis 3.4 software was used for data analysis. The respective proteins were searched by MASCOT program (Matrix Science, London, England) and identification was performed using the NCBI-nr protein sequence database (NCBI, Bethesda, MD, USA) which used MOWSE algorithm.
The data were screened against the NCBI-nr database with the help of the following parameters: taxonomy—Oryza sativa (25805290 sequences); cleavage specificity—trypsin with one missed cleavages allowed; allowed modifications—carbamidomethyl (fixed), oxidation of methionine (variable); cleavage by trypsin—cuts C-terminus side of KR unless the next residue is P. Only the significant hits (P < 0.05) were taken on the basis of MASCOT probability analysis.