Computational protocol: Overexpression of the Vitronectin V10 Subunit in Patients with Nonalcoholic Steatohepatitis: Implications for Noninvasive Diagnosis of NASH

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Protocol publication

[…] An UltiMate 3000 RSLC nano-LC system (ThermoFisher Scientific, Waltham, MA, USA) equipped with an integrated nanoflow manager and microvacuum degasser was used for peptide separation. The peptides were loaded onto a 75 μm NanoSeries C18 column (ThermoFisher, P/N 164534) for multistep gradient elution (eluent A 0.05% TFA; eluent B 0.04% TFA in 80% ACN) from 5% to 20% eluent B within 10 min, from 20% to 50% eluent B within 45 min, and for further 5 min from 50% to 90% eluent B with a constant flow of 0.3 μL/min. After 5 min, the eluted sample fractions were continuously diluted with 1.2 μL/min a-cyano-4-hydroxycinnamic acid (CHCA) and spotted onto a MALDI target using a HTC-xt spotter (PAL SYSTEM) with an interval of 20 s resulting in 168 fractions for each gel slice. Mass Spectrometry Analysis MALDI-TOF MS spectra were acquired using a 5800 MALDI TOF/TOF Analyzer (Sciex, Concord, ON, Canada). The spectra were acquired in the positive reflector mode by 20 subspectral accumulations (each consisting of 50 laser shots) in an 800−4000 mass range, focus mass 2100 Da, using a 355 nm Nb:YAG laser with a 20 kV acceleration voltage. Peak labeling was automatically done by 4000 Series Explorer software Version 4.1.0 (Sciex) without any kind of smoothing of peaks or baseline, considering only peaks that exceeded a signal-to-noise ratio of 10 (local noise window 200 m/z) and a half maximal width of 2.9 bins. The calibration was performed using default calibration originated by five standard spots (Mass Standards kit for Calibration P/N 4333604). Only the MS/MS spectra of preselected peaks (out of peak pairs with a mass difference of 6.02, 10.01, 12.04, 16.03, and 20.02 Da) were integrated over 1000 laser shots in the 1 kV positive ion mode with the metastable suppressor turned on. Air at the medium gas pressure setting (1.25 × 10−6 Torr) was used as the collision gas in the CID-off mode. After smoothing and baseline subtractions, spectra were generated automatically by 4000 Series Explorer software. The MS and MS/MS spectra were processed by ProteinPilot Software 4.5 (Sciex) which acts as an interface between the Oracle database containing raw spectra and a local copy of the MASCOT search engine (Version 2.1, Matrix Science, Ltd.). The Paragon algorithm was used with identification as the Sample Type, iodacetamide as cysteine alkylation, with the search option “biological modifications” checked, and trypsin as the selected enzyme. MS/MS protein identification was performed against the Swiss-Prot database (number of protein sequences: 254757; released on 20121210) without taxon restriction using a confidence threshold of 95% (Proteinpilot Unused score ≥ 1.31). The monoisotopic precursor ion tolerance was set to 0.12 Da and the MS/MS ion tolerance to 0.3 Da. The minimum required peptide length was set to six amino acids. […]

Pipeline specifications

Software tools ProteinPilot, Mascot Server
Application MS-based untargeted proteomics
Organisms Homo sapiens
Diseases Brain Diseases, Fatty Liver, Carcinoma, Hepatocellular, Liver Cirrhosis, Non-alcoholic Fatty Liver Disease