| Metadata |
| datasetIdentifier | PASS01510 |
| datasetType | MSMS |
| submitter | Yong Chi <ychi@fredhutch.org> |
| submitter_organization | Fred Hutchinson Cancer Research Center |
| lab_head_full_name | Bruce E Clurman |
| lab_head_email | bclurman@fredhutch.org |
| lab_head_organization | Fred Hutchinson Cancer Research Center |
| lab_head_country | USA |
| datasetTag | YChi_Jan2020 |
| datasetTitle | Ychi_2020_A novel landscape of nuclear human CDK2 substrates revealed by in situ phosphorylation |
| publicReleaseDate | 2020-04-19 00:00:00 |
| finalizedDate | 2020-01-20 12:42:43 |
| summary | Identification of human CDK2 substrates by nuclei in situ labeling using ATP analog and mass spectrometry. |
| contributors | Yong Chi, John H Carter, Jherek Swanger, Alexander V Mazin, Robert L Moritz, Bruce E Clurman |
| publication | Chi, Y, Carter JH, Swanger, J, Mazin, AV, Moritz, RL, Clurman, BE, A novel landscape of nuclear human CDK2 substrates revealed by in situ phosphorylation, Science Advances, submitted. |
| growth | HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, 11965-092; Gibco), which was supplemented with 10% (v/v) of fetal bovine serum (FBS) (10437-028; Gibco), 100 U/ml penicillin, and 100 µg/ml streptomycin (15140-122; Gibco). |
| treatment | Roscovitine (R7772; Sigma) was used at 25 μM for 1 h. |
| extraction | HEK293A cells stably expressing WT-CDK2 or AS-CDK2 were grown to ~80-90% confluence, treated with roscovitine for 1 h, and harvested by trypsinization. Cell nuclei were isolated by hypotonic lysis and sucrose centrifugation. |
| separation | Thiophosphopeptides from the peptide mixture were purified by binding to 40 microliter of disulfide beads Thiopropyl Sepharose 6B (17042001; GE Healthcare) at pH 4.0 as described previously (Chi et al., 2008). Washed beads were eluted with 30 microliter of 25 mM DTT (pH = ~4 without buffering) in 5% acetonitrile/95% H2O at RT for 30 min. The eluate was acidified with tris(2-carboxyethyl)phosphine (TCEP) and formic acid to a final concentration of 5 mM and 0.1%, respectively, and analysed directly by MS. |
| digestion | The supernatant was digested with sequencing grade modified trypsin (Promega) at 1:20 ratio (w/w). |
| acquisition | Phosphopeptides samples were analysed by Nanoflow liquid chromatography (NanoLC) and electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using an LTQ-Orbitrap mass spectrometer (ThermoFisher Scientific) interfaced with an Agilent 1100 nano pump with electronically controlled split flow. For ATP-γ-S labelling, one sample was analysed in duplicate MS runs, and for PE-ATP-γ-S labelling, eight samples (4 WT-CDK2 and 4 AS-CDK2) were analyzed in duplicate MS runs (16 MS runs total). Peptides were loaded in sequence onto a 75 mirometer (I.D.) x 15 cm C18 microcapillary column, packed in house with Magic C18 AQ 5 micrometer resin (Michrom Bioresources), and resolved by a non-linear gradient of 5%-28% acetonitrile containing 0.1% formic acid at a flow rate of 300 nl/min over the course of 80 min. Each survey scan in the Orbitrap was followed by MS/MS scans of the top 9 most intense precursor ions in the linear ion-trap. |
| informatics | Tandem spectra acquired were searched against a human Uniprot database (downloaded 01/2015) with target-decoy using the Comet algorithm (version 2014.02) Peptide search parameters included precursor mass tolerance of 20 ppm, one tryptic end for peptide, and differential mass modification to methionine (+15.999) due to oxidation and serine and threonine (+96.0329) due to thiophosphorylation. Search results were filtered using Trans Proteomic Pipeline with a minimal iProphet score of 0.75 and corresponding peptide false discovery rate (FDR) between 0.5-1%. |
| instruments | Thermo Scientific LTQ Orbitrap |
| species | Human |
| massModifications | variable: M+15.9949, ST+95.943487 |
