Computational protocol: Isotope Coded Protein Labeling analysis of plasma specimens from acute severe dengue fever patients

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Protocol publication

[…] The ICPLTM technique allows the comparative analysis of complex samples by an isotopic labeling of the intact proteins. ICPLTM and protein identification have been conducted by InnovaProteomic (Rennes, France). Before ICPLTM-labeling, acetone precipitation of proteins from either DF or DHF depleted pool of plasma was carried out. To one volume of sample 5 volumes of acetone cooled to −20°C were added and incubated overnight at −20°C. Precipitated proteins were pelleted by centrifugation 15 min at 15,000 g at 4°C. Pellets were washed with cold 80% (v/v) acetone and dried. The protein concentration, previously determined by a Bradford assay, was used to adjust sample concentration at 3 mg/ml in ICPLTM lysis buffer containing 6 M Guanidine-HCl pH8.5 (SERVA, ICPLTM Kit). For equal amounts of proteins (60 μg for each sample), cysteines were alkylated by addition of 0.4 M of iodoacetamide and differentially labeled using the ICPLTM kit containing 12C- (light labeling) and 13C6–Nicotinoyloxysuccinimide (heavy labeling) from Serva Electrophoresis GmbH (Heidelberg, Germany) as described by the manufacturer []. Thereafter the two ICPLTM labeled samples were combined and proteins were precipitated by acetone to remove excess labeling reagent. Proteins were solubilized in Laemmli sample buffer, separated on 12% SDS-PAGE (GeBa, Interchim) in duplicate and stained with EZblue (Sigma Aldrich, Saint Louis, USA). After staining, the gel lanes were cut into 20 pieces and in-gel digestion by Trypsin was performed overnight at 37°C, according to the conventional method []. Finally, the peptides were extracted with 50% (v/v) acetonitrile containing 0.1% of formic acid []. The extracts were evaporated using a vacuum concentrator. The dried peptide samples were stored at −80°C until analysis.Peptide separation was done by liquid chromatography using a nanoLC Ultimate system (Dionex GmbH, Idstein, Germany) coupled to an ion trap Esquire HCT ultra (Bruker Daltonics GmbH, Bremen, Germany). Mass spectra were recorded and the quantification was operated by the WARP-LC 1.1 software. To identify proteins, searches against the MSDB database (version 2007, 148 210 sequences for human) using Mascot 2.3 software (Matrix Science, in-house server) were carried out via Biotools software (Bruckner Daltonic). Proteins were identified with a score higher than 36 (p<0.05).In order to eliminate false matches and incorrect protein identification, search on the SwissProt-Trembl_Decoy database was performed using the Mascot 2.3 software. The IRMa software [] was used to filter the results so that the rate of false positives was lower than 1%.The change in protein abundance was expressed by the calculation of a ratio between the two type of labeled peptides (heavy and light). To calculate the heavy/light (H/L) ratios of a protein at least two unique peptides labeled with heavy and light reagents were chosen respectively and a global average ratio was determined. For each result, standard deviation was calculated.Three independent analytical replicates were performed for each 1D-gel band. [...] Protein quantities were measured twice in duplicate on each DF or SD plasma specimens using commercial ELISA. Human Ferritin and LRG1 kits were purchased from IBL international (Hamburg, Germany) and the Human Vitamin D binding–protein from USCN Inc. (Wuhan, China). Protocols were performed as recommended by the manufacturers. For each test a standard curve was established with serial dilution of calibrator in order to determine the protein concentration. Statistical analysis were performed using the Student t test and the GraphPadPrism 4.03 software. […]

Pipeline specifications

Software tools Biotools, GraphPad Prism
Applications Miscellaneous, Population genetic analysis
Organisms Homo sapiens
Diseases Dengue, Virus Diseases
Chemicals Vitamin D