Ultimate 3000 rslcnano uplc system

Chromatographic separation was performed using a Dionex UltiMate 3000 RSLCnano UPLC system coupled to the nanospray ion source containing a capillary column (75 μm ID, 15 cm length, 10 μm orifice) created by hand packing a commercially available fused-silica emitter (New Objective, Woburn, MA) with XBridge BEH C18 (5 μm particle size, 130 Å pore size) stationary phase (Waters, Milford, MA). The LC solvents were: (A) 0.05% CH₃COOH in H₂O and (B) CH₃CN. Three μL injections were performed and the initial flow rate and composition of 5% B and 600nl/min was held for 10 min to push sample out of the 5 μL autosampler loop, followed by switching the injection valve from the inject to load position to take the loop out of the flow path, and a decrease of the flow rate to 300 nL/min over the course of 1 min. This was followed by a linear gradient of the B composition over 6.5 min to 61.5% B at which point the flow rate was decreased to 150 nL/min and held at 61.5% B for 2 min. The column was re-equilibrated between injections at the initial conditions for 9 min. To assure maximum capture of the 3 μL injection from the 3 μL sample volume, the sampler sample height was set to 0.0 mm in the Xcalibur method and the sample container height offset was set to 0.0 mm in the DCMSLink Chromeleon Xpress UPLC control software.

One forward and two reverse SILAC (stable isotope labeling by amino acids in cell culture) experiments were conducted [12 (link)]. The lysates of heavy-labeled ALKBH4-knockout cells and light-labeled parental HEK293T cells were mixed at a 1:1 ratio (by mass, based on quantification using the Bradford assay) in the forward labeling experiment. The reverse labeling experiment was performed in the opposite way. The lysate mixtures were subsequently loaded onto a 10% SDS-PAGE gel and ran at 80 V for 2 h. The gel lanes were cut into 10 slices based on apparent molecular weights of proteins, and these gel slices were cut into ~1 mm³ cubes. Proteins were digested in-gel, at 37°C overnight, with MS-grade trypsin (Pierce) at an enzyme/substrate ratio of 1:100 in 25 mM ammonium bicarbonate in H₂O/CH₃CN (9/1 v/v) [13 (link)]. After elution from the gel, the peptide fractions were dried in a Speed-vac and desalted with OMIX C18 Tips (Agilent Technologies). The samples were analyzed by LC-MS/MS in a DDA mode on a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Scientific, CA) coupled with a Dionex UltiMate 3000 RSLCnano UPLC system. Maxquant, Version 1.5.2.834, was utilized to identify the light- and heavy-labeled peptides and quantify their intensity ratios, and UniProt database (UP000005640) was used for the database search.