Proteomic profiling reveals ACSS2 facilitating metabolic support in Acute Myeloid Leukemia _Supplement_dataset

Mass Spectrometry and data preprocessing for phosphoproteomics of KG1a, Hs5, and KG1a-Hs5 

Samples were analyzed using a QExactive mass spectrometer (Thermo Scientific) and a U3000 RSLC nano HPLC system (Dionex) coupled to a 50 cm-long and 75-micron internal diameter EASYspray column. Samples were resolved using a 4 h gradient with the mass spectrometer run in data-dependent analysis mode, in which the 20 most intense multiply charged precursors were selected for fragmentation. The mass spectrometer settings were as follows: Precursor resolution; 70000, AGC target; 3000000, maximum fill time; 250 ms, MSMS resolution; 17500, AGC target 100000' maximum fill time was 120 ms, isolation window; 3 Da, normalized collision energy (NCE); 30, underfill ratio 10%. Data were processed and quantified using Proteome Discoverer 1.4 (Thermo Scientific) and Mascot (MatrixScience). Search parameters in Mascot (via PD) were as follows – Enzyme (trypsin), Dynamic modifications: Oxidation (M), Phosphorylation (STY), Dimethyl light, intermediate or heavy of (K) and (N-term); Static modifications: Carbamidomethyl (C); Precursor Mass tolerance: 10 ppm; Fragment Mass tolerance 0.02 Da; Missed Cleavages: 2. Data were searched against the UniProt Homo sapiens reference proteome version 2.5 (retrieved on 08/12/2013). Site localization probabilities were calculated by PhosphoRS 3.154, which is implemented in Proteome Discoverer. The statistical programming language R (https://www.r-project.org) was used for subsequent data analysis and visualization. Peptide data were extracted from Proteome Discoverer and analyzed using an in-house R script. Peptide-spectrum matches were filtered at a 1% False Discovery Rate (sequence identification); the list of phosphorylated peptides was then further filtered to have a site localization score of at least 0.75. Phosphosite abundances were transformed to log2 scale and normalized to mean 0 and standard deviation 2 separately for each time point and each measurement.  Processed phosphoproteomic data can be accessed at 10.6084/m9.figshare.23261309.