Computational protocol: Decellularization and Solubilization of Porcine Liver for Use as a Substrate for Porcine Hepatocyte Culture

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Protocol publication

[…] Proteins extracted from the pLECM (see Protein Extraction and Analysis section) were separated by 1-D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a Criterion XT 12% gel and then stained with Coomassie blue. The protein-containing region of each gel lane was divided into 7 slices which were individually reduced in situ with tris (2-carboxyethyl) phosphine and alkylated in the dark with iodoacetamide prior to treatment with trypsin (Promega, V5111, Madison, WI, USA). The digests were analyzed by capillary high-performance liquid chromatography (HPLC)-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) on a Thermo Fisher LTQ Orbitrap Velos Pro mass spectrometer fitted with a New Objective Digital PicoView 550 NanoESI source. Online HPLC separation of the digests was accomplished with an Eksigent/AB Sciex NanoLC-Ultra 2-D HPLC system (column, PicoFrit™ [New Objective; 75 µm internal diameter]) packed to 15 cm with C18 adsorbent (Vydac, Columbia, MD, USA; 218MS 5 µm, 300 Å). Precursor ions were acquired in the Orbitrap from 300 to 2,000 m/z in profile mode at 60,000 resolutions (400 m/z); data-dependent collision-induced dissociation spectra of the 6 most intense ions in each precursor scan were acquired at the same time in the linear trap (MS2 isolation width = 3; unassigned charge states rejected; normalized collision energy = 30; dynamic exclusion repeat count = 1; dynamic exclusion repeat duration = 30 s). Mascot (Matrix Science, Boston, MA, USA) was used to search the spectra against the mammal’s subset of the National Center for Biotechnology Information (NCBI) nr database. Cysteine carbamidomethylation was set as a fixed modification, and methionine oxidation and deamidation of glutamine and asparagine were considered as variable modifications; trypsin was specified as the proteolytic enzyme, with 1 missed cleavage allowed. The Mascot results files were combined for subset searching of the identified proteins by X! Tandem cross-correlation with the Mascot results, and determination of protein and peptide identity probabilities was accomplished by Scaffold (Proteome Software, Inc., Portland, OR, USA). The thresholds for acceptance of peptide and protein assignments in Scaffold were 95% and 99%, respectively, 2 peptides minimum per protein. [...] Scaffold (Proteome Software) was used to validate protein identifications derived from MS/MS sequencing results using the X! Tandem and ProteinProphet computer algorithms. Scaffold verified peptide identifications assigned by SEQUEST, Mascot, or other search engines (NCBI, Pantherdb) using the X! Tandem database searching program, Scaffold then probabilistically validated these peptide identifications and derived corresponding protein probabilities using ProteinProphet.– All protein sequences were filtered by taxonomy (Sus scrofa). Common and statistically different proteins were analyzed in protein analysis through evolutionary relationships. Proteins were classified using comprehensive gene ontology (GO), annotations. Details of the methods can be found elsewhere., […]

Pipeline specifications

Software tools X! Tandem, ProteinProphet, Comet, PANTHER
Applications MS-based untargeted proteomics, Protein sequence analysis
Organisms Homo sapiens
Chemicals Hydrochloric Acid, Sodium, Urea, Acetic Acid, Ammonium Hydroxide