Proteomic Analysis of Human Liver S9 Fraction by 2D-LC-MSMS
Liver plays a crucial role in numerous metabolic processes and therefore is the primary target of study in drug discovery and molecular toxicology. Devising an appropriate analytical strategy for an efficient analysis of the liver samples has been a major concern in proteomics. The aim of this study was to develop an LC-MS based analytical method for identification of liver proteins with adequate peptide-level confidence and protein sequence coverage. The present dataset will improve the existing knowledge on the proteins identified in human liver S9 fraction.
Sample Processing Protocol
Two human liver S9 fraction (HLS) samples were solubilized in 2% SDS; one was digested by trypsin (HST) while the other was subjected to a pepsin digestion (HSP). Digest peptides from each sample were cleaned up using solid-phase extraction (SPE) and fractionated into six fractions by strong cation exchange (SCX) prior to an ultra-high pressure reverse-phase chromatography (RP-UHPLC) directly coupled to tandem mass spectrometer. All MS and MS/MS spectra were collected on a high-resolution hybrid quadrupole-time of flight TripleTOF 5600 mass spectrometer equipped with a DuoSpray ion source in positive ion mode. The instrument performed a survey TOF-MS acquisition from m/z 140–1250 with an accumulation time of 250 ms, followed by MS/MS on the fifteen most intense ions from m/z 80–1500 with exclusion after two occurrences for 20 s using information-dependent acquisition (IDA) with dynamic background subtraction (DBS). Each MS/MS had an accumulation time of 50 ms and collision energy ranging from 20 to 40 V, depending on the charge state and mass of the product ion. The total cycle time was 1.05 s. All samples were prepared in duplicates.
Data Processing Protocol
MS/MS files from the tryptic and peptic digests (HST and HSP) were combined and searched against UniProt protein database (release date 26/06/2013) by ProteinPilot software (version 4.1) using Paragon algorithm. The ID focus was on biological modifications, including false discovery rate analysis and detection protein threshold of unused ProtScore > 0.05 (confidence >10%). The search was performed for +2 to +4 charge states and MS tolerance was 0.05 Da on precursor ions and 0.1 Da on fragment ions using ProteinPilot’s default workflow directory.
Lekha Sleno, Université du Québec à Montréal (UQÀM), Chemistry Department / Pharmaqam, Montréal, QC, Canada
Lekha Sleno, Bioanalytical Mass Spectrometry Research Group, Chemistry Department, Universite du Quebec a Montreal (UQAM), Montreal (Quebec), Canada ( lab head )
Golizeh M, Schneider C, Ohlund LB, Sleno L. Dataset from proteomic analysis of rat, mouse, and human liver microsomes and S9 fractions. Data Brief. 2015 Feb 24;3:95-8. eCollection 2015 Jun PubMed: 26217725