Project description:The following cell lines were used for this study: Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs) derived from 5 human (Coriell YRI, NIGMS Human Genetic Cell Repository, GM18505, GM18507, GM18516, GM19193, GM19204), and 5 chimpanzee (Pan troglodytes) individuals (New Iberia Research Center: Min 18358, Min 18359; Coriell/IPBIR: NS03659, NS04973, Arizona State University, Pt91), and rhesus Herpesvirus papio transformed LCLs from 5 rhesus macaque (Macaca mulatta) individuals (Harvard Medical School, NEPRC: 150-99, R181-96, R249-97, 265-95, R290-96). Cells were maintained at identical conditions of 37° with 5% CO2 in RPMI media with 15% FBS, supplemented with 2 mM L-glutamate, 100 IU/ml penicillin, and 100 ug/ml streptomycin. Internal standard LCL (Coriell YRI, NIGMS Human Genetic Cell Repository, GM19238) was grown in RPMI minus L-Lysine and L-Arginine, 15% dialyzed FBS, and L-13C615N4-arginine (Arg-10) and L-13C615N2-lysine (Lys-8) (Cambridge Isotopes, Andover, MA, USA) supplemented with 2 mM L-glutamate, 100 IU/ml penicillin, and 100 ug/ml streptomycin under identical conditions as the unlabeled LCLs. The internal standard LCL was grown for 6 doublings to assure complete SILAC label incorporation. Complete label incorporation was verified by analyzing the protein lysate from the labeled LCL alone by high-resolution LC-MS/MS. LCLs were washed in PBS three times and then lysed using the UPX Universal Protein Extraction Kit (Expedeon Inc., San Diego, CA, USA). Protein quantitation was performed using the Qubit fluorometry assay (Invitrogen, Carlsbad California, USA) and the reducing agent-compatible (RAC) version of the BCA Protein Assay (Thermo Scientific, Waltham, Massachusetts, USA). 12ug of each sample was combined with 12ug of the SILAC labeled lysate from human LCL GM19238. Note that the SILAC lysate was prepared once and used as an internal standard through the quantification of the 15 cell lines. 24ug of each combined sample was then processed by SDS-PAGE using a 4-12% Bis Tris NuPage mini-gel (Invitrogen, Carlsbad California, USA). Calibration was with Thermo PageRuler broad range markers. Each of 40 gel segments were processed by in-gel digestion using a ProGest robot (DigiLab, Marlborough, MA, USA) with the following protocol: wash with 25mM ammonium bicarbonate followed by acetonitrile, reduce with 10mM dithiothreitol at 60°C followed by alkylation with 50mM iodoacetamide at room temperature, digest with trypsin (Promega, Madison, WI, USA) at 37°C for 4h, and quench with formic acid. The supernatant was analyzed directly without further processing. Each of gel digest was analyzed by nano-LC/MS/MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher LTQ-Orbitrap Velos Pro. Peptides were loaded on a trapping column and eluted over a 75um analytical column at 350nL/min using a 1-hour LC gradient. Both columns were packed with Jupiter Proteo resin (Phenomenex, Torrance, California, USA). The mass spectrometer was operated in data-dependent mode, with MS performed in the Orbitrap at 60,000 FWHM resolution and MS/MS performed in the LTQ. The fifteen most abundant ions were selected for MS/MS. Low-level data analysis was performed using the open-source proteomics software tool PVIEW (Release December 23, 2012; http://compbio.cs.princeton.edu/pview). As input to PVIEW, we generated in silico translations of coding genes from the UCSC Genome Browser database based on gene models from build hg19 of the human genome. Each protein sequence entry retained the corresponding Ensembl gene identifier and gene symbol. Database searches were performed using +-4 p.p.m. MS1 tolerance and an MS2 window tolerance of +-0.5 Da. Up to 2 missed tryptic cleavages were allowed during search. Carboxyamidomethylation of cysteine was used as fixed modification. Up to two methionine oxidations were allowed as variable modifications of a tryptic peptide. Peptide spectrum matches were obtained at a stringent false discovery rate (FDR) of 1%. We used the median log2(sample/standard) ratio across all independent quantifications of a protein (distinct peptides including duplicate peptide measurements across fractions and for differing charge states). Note that use of the hg19 database was sufficient as SILAC pairs as the expected isotope shift used for quantification were only present for peptides that that had the same underlying sequence in the human internal standard line. Associated RNA-seq data have been deposited to GEO with accession number GSE49682.
2013-10-01 | PXD000419 | Pride
Dmitry Alexeev 
