Equal numbers of light- and heavy-labeled HeLa cells differentially treated were mixed in a 1:1 ratio, pelleted, and washed twice with PBS. Cell pellets were lysed in urea lysis buffer [9 M urea, 20 mM Hepes (pH 8.0)] supplemented with 1× Roche proteases and phosphatase inhibitors, sonicated, and cleared by ultracentrifugation (20,000g for 15 min at 15°C). For in-solution digestion, 50 mg of proteins was reduced by adding 4.5 mM dithiothreitol (DTT) (Sigma-Aldrich) for 30 min at 55°C, alkylated with 5.5 mM iodoacetamide [10% (v/v) for 15 min at room temperature in the dark; Sigma-Aldrich], and digested overnight with sequencing-grade trypsin [1:100 (w/w); Promega] after a fourfold dilution in 25 mM ammonium bicarbonate solution. Protease digestion was terminated by the addition of trifluoroacetic acid (TFA) to adjust pH < 3. Precipitated material was removed by centrifugation for 15 min at 1780g at room temperature. Peptides were purified using reversed-phase Sep-Pak C18 cartridges (Waters, Milford, MA) and eluted off the Sep-Pak with 40% acetonitrile (ACN) with a subsequent step of removal of ACN by 48 hours of lyophilization. Lyophilized peptides were dissolved in 25 mM ammonium hydroxide (NH4OH) and subsequently offline fractionated by high-pH fractionation using a Phenomenex Jupiter C12 4 μm Proteo 90 Å, LC column 250 × 4.6 mm, on an ÄKTA-FPLC (fast protein liquid chromatography) system (GE Healthcare) operating at 1 ml/min. Buffer A was 25 mM NH4OH, and buffer B was 25 mM NH4OH in 90% ACN. Fractions were collected using a collector in a 96-deep well plate at 1-min intervals. Samples were initially loaded onto the column at 1 ml/min for 3 min, after which the fractionation gradient was as follows: 5% B to 30% B in 60 min, 30% B to 60% B in 2 min and ramped to 70% B for 3 min. At this point, fraction collection was halted, and the gradient was held at 100% B for 5 min before being ramped back to 5% B, where the column was then washed. The 60 collected fractions were concatenated to 14 throughout each experiment. After lyophilization, each fraction was dissolved in 250 μl of 1× immunoaffinity purification buffer (IAP buffer, #9993, Cell Signaling Technology) and subjected to two consecutive steps of methyl-R peptide enrichment using the SDMA antibody-conjugated beads (PTMScan [sdme-R] Kit #13563, Cell Signaling Technology) and MMA antibody-conjugated beads (PTMScan Mono-Methyl Arginine Motif [mme-RG] Kit #12235, Cell Signaling Technology) following the manufacturer’s instruction. After peptide incubation with the antibody-conjugated beads for 2 hours at 4°C, the immunoprecipitates were washed twice in ice-cold IAP buffer followed by three washes in water; then, bound methyl peptides were eluted with 2 × 50 μl of 0.15% TFA. Peptide eluates were desalted on reversed-phase C18 StageTips, as described previously (52), and subjected to a second round of trypsin digestion before nano–LC-MS/MS analysis.

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