L lysine u 13c6 15n2

HEK cells were SILAC-labeled in medium containing l-arginine and l-lysine or l-arginine-U-¹³C₆-¹⁵N₄ and l-lysine-U-¹³C₆-¹⁵N₂ (Cambridge Isotope Laboratories, Tewksbury, MA, USA) as previously described [52 (link)]. Peptides were analyzed on a quadrupole Orbitrap mass spectrometer (Q-Exactive plus, Thermo Fisher Scientific, Waltham, MA, USA) equipped with a nanoflow HPLC system (Thermo Scientific), as previously described [53 (link)]. Peptides were loaded onto C18 reversed phase columns (15 cm length, 75 μm inner diameter) and eluted with a linear gradient from 8–40% acetonitrile containing 0.5% acetic acid. The mass spectrometer was operated in a data-dependent mode, automatically switching between MS and MS/MS acquisition. Survey full scan MS spectra (m/z 300–1 750) were acquired in the Orbitrap. The 10 most intense ions were sequentially isolated and fragmented by higher-energy C-trap dissociation (HCD [54 (link)). An ion selection threshold of 50 000 counts was used. Peptides with unassigned charge states, as well as with charge state less than +2 were excluded from fragmentation. Fragment spectra were acquired in the Orbitrap mass analyzer (Thermo Fisher Scientific).

HeLa cells (ATCC) were cultivated in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum and antibiotics. Cells were synchronized by thymidine (2.5 mM) the day before co-transfection with siRNA oligos against Bub1 (100 nM as final concentration) and corresponding plasmids by Lipofectamine 2000 (Life Technologies). RNAi oligos targeting Bub1 (5′-GAGUGAUCACGAUUUCUAA-3′) or luciferase (5′-CGUACGCGGAAUACUUCGA-3′), were used for RNAi depletions. Thymidine was added again 12 h later after transfection. A second RNAi was performed during the second thymidine block. Twenty four hours later, the cells were released from thymidine block. Filming or fixation was performed when cells entered mitosis.
For SILAC labelling, HeLa cells were grown in SILAC DMEM medium (Invitrogen) supplemented with 10% dialysed FBS, L-glutamine, penicillin/streptomycin and isotope labelled arginine and lysine. L-lysine and L-arginine (Sigma) were used for light culture. L-lysine 4,4,5,5-D4 and L-arginine–U-13C6, or L-lysine-U-13C6-15N2 and L-arginine–U-13C6-15N4 (Cambridge Isotope Laboratories) were used for medium or heavy culture.