MassIVE MSV000093903

Complete Public PXD048773

Phosphoproteomic Analysis of PCB126 Impact on the Mouse Chronic-Binge Alcohol Feeding Model

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Description

Environmental pollutants, including polychlorinated biphenyls (PCBs) have been implicated in the pathogenesis of liver disease. To better understand how PCB126 promoted alcohol-associated liver disease (ALD) in our previous model, the current study utilized a phosphoproteomic approach for hypothesis generation regarding mechanisms of action. Briefly, male C57B/6J mice were exposed to 0.2 mg/kg polychlorinated biphenyl PCB126 or corn oil vehicle prior to ethanol (EtOH) or control diet feeding in the chronic-binge alcohol feeding model. Liver tissues were harvested during euthanasia and tissue collection, snap frozen in liquid nitrogen, and then were archived in a -80C freezer. For phosphoproteome analysis, six random liver tissues from four groups (N=24) were homogenized in 2% sodium dodecyl sulfate (SDS) radioimmunoprecipitation (RIPA) buffer with 1X HALT protease and phosphatase inhibitors. Lysates were trypsinized at a 1:20 (enzyme:sample) ratio and prepared following ProtiFi S-Trap mini spin column digestion protocol. Phospho-peptides were enriched with MagReSyn titanium ion - immobilized metal affinity chromatography (Ti-IMAC) microparticles following ReSyn Biosciences MagReSyn Ti-IMAC protocol. Samples were then purified using C18 PROTO 300 A Ultra MicroSpin columns, dried under a SpeedVac, and stored at -80C prior to peptide concentration assessment. Digests were fractionated by UPLC nanoflow liquid chromatography using PepMap RSLC C18 EASY-spray separating column at 40C with a 90-minute linear gradient. Eluates were introduced into an Exploris480 using an EASY-spray source (320C and 1.8kV). Full MS-ddMS2 method with a 3 second cycle time was generated in Xcalibur (v4.5.445.18) operating in positive polarity. RAW data files were separately searched in PeaksXpro (Bioinformatics Solutions Inc.; Waterloo, ON, Canada) using Denovo, PeaksDB, and PeaksPTM algorithms against the UniprotKB Mus musculus canonical protein sequences (proteomics ID: UP000000589) downloaded March 17, 2023. Label Free Quantification algorithm was then used with PeaksPTM results utilizing a mass error tolerance of 10 ppm and retention time shift tolerance of 6-minutes. Peptides were accepted at a 1% FDR threshold for consideration by the Label Free Quantification algorithm. [doi:10.25345/C5SX64M6V] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: liver ; PCB ; binge ethanol ; chronic ethanol

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Principal Investigators:
(in alphabetical order) 		Timothy D. Cummins, PhD, University of Louisville, USA
Submitting User: mmerchant
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