Ltq orbitrap xl linear ion trap mass spectrometer

A Coomassie-stained SDS-PAGE gel band containing the YBX1 protein was subjected to in-gel tryptic digestion as previously described^{36 (link)}. Following destaining procedures (50% acetonitrile in 100 mm ammonium bicarbonate and 100% acetonitrile), cysteine residues were reduced using 20 mm DTT at room temperature for 60 min. Alkylation of the sample was then performed with 55 mm iodoacetamide, 30 min in the dark. Gel pieces were then washed with 100 mm ammonium bicarbonate, dehydrated in acetonitrile and dried in a SpeedVac centrifuge. Trypsin (Promega, WI) was used to rehydrate and digest the sample, overnight at 37 °C. Extracted peptides were reconstituted in 0.1% formic acid and prepared for mass spectrometry analysis. Proteolytic digests were analyzed using an LTQ Orbitrap XL linear ion-trap mass spectrometer (Thermo Fisher Scientific) coupled with an Ultimate 3000 HPLC system (Dionex). The data were analyzed by Mascot software (Matrix Science) against customized YBX1 protein database with the setting of 10 ppm for precursor ions and 0.8 Da for product ions. The tandem mass spectra of candidate-modified peptides were further interpreted manually.

The eluted immune complexes were precipitated with 20% trichloroacetic acid and the pellets were washed four times with cold acetone. The precipitated proteins were resuspended in 100 mM ammonium bicarbonate (pH 8.0) with 10% acetonitrile and incubated with sequencing-grade Trypsin (Promega, Fitchburg, WI, USA) at a concentration of 12.5 ng ml⁻¹ at 37 °C for 4 h. Trypsin reactions were quenched by addition of 5% formic acid, and peptides were desalted using the C₁₈ Stage Tip method. For each liquid chromatography–MS/MS analysis, 4 μl of sample was loaded onto an EASY-Spray C₁₈ column (Thermo Scientific, Waltham, MA, USA) and eluted using a 90-min 8–26% acetonitrile gradient. Mass spectra were acquired with an LTQ Orbitrap XL linear ion trap mass spectrometer (Thermo Scientific) using a data-dependent Top-10 method. Each sample was shot twice in succession, followed by a wash with 70% acetonitrile and 30% isopropanol. MS/MS data were analysed using the Coon OMSSA Proteomics Software Suite41 (link). Z-score is representative of the identification of candidate interacting proteins. Total Spectral Count (TSC) is for each identified protein from each immunoprecipitation–MS/MS experiment.