Computational protocol: Vitamin K epoxide reductase regulation of androgen receptor activity

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Protocol publication

[…] RNA sequencing was performed by the City of Hope Integrative Genomics core facility. cDNA synthesis and library preparation was performed using TruSeq RNA Library prep kit in accordance with the manufacturer supplied protocols. Libraries were sequenced on the Illumina Hiseq 2500 with single read 40 bp reads. The 40-bp long single-ended sequence reads were mapped to the human genome (hg19) using TopHat and the frequency of Refseq genes was counted with customized R scripts. The raw counts were then normalized using trimmed mean of M values (TMM) method and compared using Bioconductor package “edgeR”. The average coverage for each gene was calculated using the normalized read counts from “edgeR”. Differentially regulated genes were identified using one-way ANOVA with linear contrasts to calculate p-values, and genes were only considered if the false discovery rate (FDR) was < 0.25 and the absolute value of the fold change was > 1.5. Gene ontology analyses were performed using GSEA [], DAVID [] and Ingenuity Pathway Analysis (Qiagen). [...] Immunoprecipitated AR was separated by SDS-PAGE and stained with SimplyBlue (Themofisher). Gel bands were excised and destained in ammonium bicarbonate. After disulfide bond reduction with 10 mM tris(carboxyethyl)phosphine and thiol alkylation with 50 mM iodoacetamide, gel bands were incubated with trypsin/Lys-C or chymotrypsin (Promega) overnight. Peptides were extracted with 0.1% TFA/70% acetonitrile and lyophilized. Methyl esterification was performed by incubating in 2M methanolic HCl for 1 hour at 20°C to improve detection of γ-carboxyl groups []. Samples were lyophilized again and resuspended in 0.1% formic acid. Mass spectrometric analyses of the digest peptides were conducted on an Orbitrap Fusion hybrid mass spectrometer (Thermo Fisher) equipped with an Easynano UHPLC, using a 75 μm × 250 mm Pepmap RSLC reverse phase column with a PepMap 1000 trapping column (Thermo Fisher). 10 μl of methylated peptide samples were loaded at 4 μl per minute. LC was performed with a gradient mobile phase system containing buffer A (0.1% formic acid) and buffer B (100% acetonitrile/0.1% formic acid). A 40 minute gradient elution from the analytical column was conducted from 3% to 80% buffer B, followed by 45–60 minutes at 90% solvent B. Flow rate was 300 nl/min. Full mass scans (200–4000 Da) were taken by the Orbitrap mass analyzer, operated at 120K resolution, while collision-induced dissociation (CID) was conducted in data-dependent mode to generate MS/MS data. The data was analyzed using PEAKS Studio (Bioinformatics Solutions Inc.) and Proteome Discoverer (Thermo Fisher) using a non-redundant human protein database (Swissprot and NR) with the tagged AR sequence added. Database searches were carried out by considering three missed enzymatic cleavages, a precursor ion mass tolerance of 5 ppm and 0.02 Da mass tolerance for fragment ions. Search parameters also included cysteine carbamidomethylation, methionine oxidation, glutamate carboxylation, and carboxy methyl esters were searched as expected amino acid modifications. […]

Pipeline specifications

Software tools PEAKS X, Proteome Discoverer
Application MS-based untargeted proteomics
Organisms Mus musculus
Diseases Blood Coagulation Disorders, Prostatic Neoplasms
Chemicals Vitamin K, Warfarin, Glutamic Acid