Computational protocol: DNA Damage Regulates Translation through β-TRCP Targeting of CReP

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Protocol publication

[…] The immunopurified protein complexes were mixed in a ratio of 1:1 with digestion buffer (100 mM Tris-HCl, pH 8.5, 8M urea), reduced, alkylated and digested by sequential addition of lys-C and trypsin proteases as previously described[,]. For identification of phosphorylation site, proteins were digested directly in the excised gel slice using trypsin[]. Peptide digests desalted and fractionated online using a 50 μM inner diameter fritted fused silica capillary column with a 5 μM pulled electrospray tip and packed in-house with 15 cm of Luna C18(2) 3 μM reversed phase particles. The gradient was delivered by an easy-nLC 1000 ultra high pressure chromatography system (Thermo Scientific). MS/MS spectra were collected on a Q-Exactive mass spectrometer (Thermo Scientific) [,]. Data analysis was performed using the ProLuCID, DTASelect2, and Ascore algorithms as implemented in the Integrated Proteomics Pipeline—IP2 (Integrated Proteomics Applications, Inc., San Diego, CA) [–]. Phosphopeptides were identified using a differential modification search that considered a mass shift of +79.9663 on serines, threonines and tyrosines. Protein and peptide identifications were filtered using DTASelect and required at least two unique peptides per protein and a peptide-level false positive rate of less than 5% as estimated by a decoy database strategy[]. Normalized spectral abundance factor (NSAF) values were calculated as described and multiplied by 105 to improve readability []. […]

Pipeline specifications

Software tools ProLuCID, DTASelect, Ascore
Application MS-based untargeted proteomics
Organisms Homo sapiens