Proteome discoverer version 1

The silver-stained protein bands were excised from the NuPAGE gels (Thermo Fisher Scientific), were cut into small pieces (approximately 1 mm³), and were destained. Proteins of the gel pieces were reduced with DTT (Thermo Fisher Scientific), were alkylated with iodoacetamide (Thermo Fisher), and were digested with trypsin and lysyl endopeptidase (Promega, Madison, Wisconsin, USA). The resultant peptides were analyzed on an Advance UHPLC system (Michrom Bioresources, Auburn, California, USA) coupled to a Q Exactive mass spectrometer (Thermo Fisher Scientific) with the raw data processed using Xcalibur (Thermo Fisher Scientific). The raw data were analyzed against the SwissProt database or NCBI nonredundant protein database restricted to Drosophila melanogaster using Proteome Discoverer version 1.4 (Thermo Fisher Scientific) with the Mascot search engine version 2.4 (Matrix Science, London, UK). A decoy database comprised of either randomized or reversed sequences in the target database was used for false discovery rate (FDR) estimation, and Percolator algorithm was used to evaluate false positives. Search results were filtered against 1% global FDR for high confidence level.

YB-1 was purified from the nuclear and cytoplasmic fractions of two cancer cell lines; the adenocarcinoma cell line A549 and the breast cancer cell line MDA-MB23, as previously described [25 (link)]. Purified YB-1 was then separated using SDS-PAGE and stained with Coomassie (Supplementary Figure S1B). Excised bands were analysed by LC-MS/MS to identify the proteins that copurified with immunoprecipitated YB-1, again as previously described [25 (link)]. Peaklists and MS/MS data were generated using the Proteome Discoverer (version 1.4) software using default settings and searched against the Human Swiss-Prot amino acid sequence database using both the Mascot (http://www.matrixscience.com) and Sequest (Thermo Scientific) search engines. The search was set up for full tryptic peptides with a maximum of 3 missed cleavage sites. Carboxyamidomethyl cysteine, oxidized methionine, deamidation (N, Q), and phosphorylation (S, T, Y) were included as variable modifications. The precursor mass tolerance threshold was 10 ppm and the maximum fragment mass error 0.8 Da. Peptides were accepted as identified if their false discovery rate adjusted scores were above the score threshold at a false discovery rate≥ 1% as assessed by the Percolator algorithm [54 (link)]. Proteins were considered identified when they were assigned ≥ 2 peptides above the aforementioned score threshold.