DOI: 10.25345/c5rj49637
title: MassIVE MSV000095760 - Hepatic proteomic changes associated with liver injury caused by alcohol consumption in Fpr2-null mice
title: The current study employed a proteomic approach to identify hepatic changes resulting from ethanol (EtOH) consumption and the genetic ablation of the formyl peptide receptor 2 (FPR2), a G-protein coupled receptor known to regulate multiple signaling pathways and biological processes, in a mouse model of ALD. Digestion and TMTPro labeling Mouse liver samples (250ug) were digested according to the S-trap protocol (Protifi,LLC, Fairport, NY, USA). Peptide recovery estimated by a pierce quantitative colorimetric peptide assay (ThermoFisher Scientific, Waltham, MA, USA). Liver digests (100ug) were labeled with TMTpro 16Plex (ThermoFisher) reagents, admixed into a single pool, then desalted using an Oasis HLB 1cc (30mg) Cartridge (Waters Corporation, Milford, MA, USA). First dimension fractionation Samples were fractionated using a U3000SD uHPLC system (ThermoFisher Scientific) and a Waters BEH XBridge C18 5um 3.0 x 150mm column. Fractions were collected in 1.25min intervals over a gradient of solvents A = 5% v/v acetonitrile / 10mM ammonium formate pH10 and B = 90% v/v acetonitrile / 10mM ammonium formate pH10 (0% to 40% B over 65min at a flow rate of 300uL/min). A total of 54 collected fractions were concatenated into 18 by pooling three fractions (fractions n, n+18, n+36) plus the final fraction collected from the end of the LC separation. LC-MS data collection The fractions were dried in a SpeedVac and submitted for LC-MS analysis at the University of California Davis Genome Center Proteomics Core Facility. In brief samples were separated on a RSLCNano system at 500nL/min with a 70min gradient from 3% to 55% acetonitrile/0.05% formic acid gradient. Eluates were introduced into an Orbitrap Fusion Lumos mass spectrometer (ThermoFisher) system with ion transfer capillary temperature and spray voltage of the mass spectrometer set at 275oC and 1.8kV, respectively. The MS was operating in positive polarity and collected data using a TMT SPS MS3 method with a 3 second cycle time. An Orbitrap MS1 master scan (normal mass range; 120,000 resolution, AGC target 4E5, profile data type) was obtained using the range 400-1400m/z. MS2 scans (rapid scan rate, AGC target 2E4, centroid data type) were obtained in the ion trap on peaks that had a minimum signal threshold of 5,000 counts. Multi-notch (10 notches) Orbitrap MS3 scans (50,000 resolution, AGC target 1E5, centroid data type) were obtained using the range 100-500m/z. Data Analysis The collected data analysis was directed through Proteome Discoverer v2.4.0.305 (ThermoFisher) using SequestHT and the 1/29/2021 version of the UniprotKB reviewed canonical Mus musculus sequences (Proteome ID UP000000589) considering trypsin (KR|P) digestion with up to two missed cleavages, dynamic modifications Oxidation (M), Acetyl (Protein N-term), Met-los (Protein N-term), and Met-loss+Acetyl (Protein N-term); and the static modifications Carbamidomethyl (C) and TMTpro (K, any N-term). Precursor and fragment mass tolerances were 10ppm and 0.6Da, respectively. In the consensus step, proteins were quantified from the summed abundances of all high confidence unique and razor reporter ion abundances. Samples were normalized to total peptide amount and scaled to 100 on all average. Proteins were grouped by the strict parsimony principle. Peptides and proteins were accepted at 1% FDR for high confidence or 5% for medium confidence based on the q-value. A proteins text file was exported from the consensus workflow result of Proteome Discoverer for curation in Microsoft Excel.