Proteomic analysis of "Defects in a liver-bone axis contribute to hepatic osteodystrophy disease"
Description
LC-MS/MS Analysis and data analysis. Data acquisition was performed with a Triple TOF 5600 System (AB SCIEX, Concord, ON). Samples were chromatographed using a 90 min gradient from 2-30% (mobile phase A 0.1% (v/v) formic acid, 5% (v/v) acetonitrile; mobile phase B 0.1% (v/v) formic acid, 95% (v/v) acetonitrile) after direct injection onto a 20 μm PicoFrit emitter (New Objective) packed to 12 cm with Magic C18 AQ 3 μm 120 Å stationary phase. MS1 spectra were collected in the range 350-1,500 m/z for 250 ms. The 20 most intense precursors with charge state 2-5 were selected for fragmentation, and MS2 spectra were collected in the range 50-2,000 m/z for 100 ms; precursor ions were excluded from reselection for 15s. The original MS/MS file data were submitted to ProteinPilot Software (version 4.5, AB Sciex) for data analysis. MS/MS data were searched against Mus musculus UniProt database (April 9, 2016, containing 50,943 sequences http://www.uniprot.org/proteomes/UP000000589). / Tandem mass spectra were searched against Homo Sapiens UniProt database (April 9, 2016, containing 160,566 sequences, http://www.uniprot.org/proteomes/UP000005640) concatenated with reverse decoy database. The following search parameters were used: the instrument was TripleTOF 5600, iTRAQ quantification, cysteine modified with MMTS; biological modifications were selected as ID focus, the Quantitate, trypsin digestion, Bias Correction and Background Correction was checked for protein quantification and normalization. For false discovery rate (FDR) calculation, an automatic decoy database search strategy was used to estimate FDR using the PSPEP (Proteomics System Performance Evaluation Pipeline Software) algorithm. Only proteins with at least two unique peptides were considered for further analysis. For protein abundance ratios measured using iTRAQ after normalization, we specifically used ratios with p value < 0.05, and only fold changes > 1.5 or < 0.667 were considered significant.