Computational protocol: Phosphorylation of LSD1 by PLK1 promotes its chromatin release during mitosis

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Protocol publication

[…] Phosphorylation site mapping by mass spectrometry was performed as described previously []. Briefly, in vitro phosphorylated proteins were precipitated with TCA and resuspended in a buffer containing 8 M urea, 100 mM Tris, pH8.5. After reduction and alkylation, the sample was digested by Trypsin overnight at 37 °C. The peptides were analyzed on an Easy-nLC 1000 UPLC (Thermo Fisher Scientific) coupled to a Q exactive mass spectrometer (Thermo Fisher Scientific). Peptides were loaded on a pre-column (75 μm ID and packed with 8 cm ODS-AQ 12 nM S-10 mm (YMC Co., Ltd)) and separated on an analytical column (75 μm ID and packed with 11 cm Luna 3 μm 100Å resin (Phenomenex)) with an acetonitrile gradient from 0 to 30% in 55 min and 30–80% in another 10 min at a flow rate of 300 nl/min. Spectra were acquired in a data-dependent mode: the 10 most intense ions except charge 1+ or unassigned from each full scan (Resolution 70,000) were isolated for HCD MS2 (Resolution 17,500) at NEC 27 with a dynamic exclusion time of 60 s. For peptide identification, the MS2 spectra were searched against an artificial database (including GST-LSD1 and HIS-PLK1 sequences in a C. elegans WS217 database) using Prolucid []. Search results were filtered using DTASelect 2.0 [] with 7 p.p.m. mass accuracy for precursor mass and a 5% FDR cutoff. The phosphorylation spectra presented in the figures were annotated using pLabel []. […]

Pipeline specifications

Software tools ProLuCID, DTASelect, pLabel
Application MS-based untargeted proteomics
Chemicals Nocodazole