Briefly, 0.2 mg of TMT-10plex isobaric labels (Thermo Scientific) per differential centrifugation fraction were equilibrated to room temperature and reconstituted in 20 µL LC-MS grade anhydrous acetonitrile. In each experiment, labels were randomly assigned to each fraction with a random number generator to mitigate possible batch effect. Isobaric tags were added to peptides still atop the centrifugation filters and incubated at room temperature for 1 hour with shaking. The reactions were quenched with 1 µL hydroxylamine at room temperature for 30 minutes with shaking. Labeled peptides were eluted from the filters with centrifugation. To further elute labeled peptides 40 µL 50 mM TEAB was added and filters were again centrifuged. All 10 labeled fractions per experiment were combined and mixed well before dividing each experiment into two aliquots.
One aliquot per experiment was reconstituted in 50 µL 20 mM ammonium formate pH 10 in LCMS grade water (solvent A) for high pH reverse phase liquid chromatography (RPLC) fractionation. The entire sample was injected into a Jupiter 4 µm Proteo 90 Å LC Column of 150 × 1 mm on a Ultimate 3000 HPLC system. The gradient was run with a flow rate of 0.1 mL/min as follows: 0–30 min: 0%–40% Solvent B (20 mM ammonium formate pH 10 in 80% LCMS grade acetonitrile); 30–40 min: 40%-80% Solvent B; 40–50 min: 80% Solvent B. Fractions were collected every minute and pooled into a total of 20 peptide fractions, then dried with speed-vac. 

The dried fractions were reconstituted in 10 µL each of pH 2 MS solvent A (0.1% formic acid) and analyzed with LC-MS/MS on a Q-Exactive HF orbitrap mass spectrometer coupled to an LC with electrospray ionization source. Peptides were separated with a PepMap RSLC C18 column 75 µm x 15 cm, 3 µm particle size (ThermoScientific) with a 90 minute gradient from 0 to 100% pH 2 MS solvent B (0.1% formic acid in 80% LCMS grade acetonitrile). Full MS scans were acquired with a 60,000 resolution. A stepped collision energy of 27, 30 and 32 was used and MS2 scans were acquired with a 60,000 resolution and an isolation window of 0.7 m/z. 
Mass spectrometry raw data were converted to mzML format using ThermoRawFileParser v.1.2.0 (Hulstaert et al., 2020) then searched against the UniProt SwissProt human canonical and isoform protein sequence database (retrieved 2022-10-27) using Comet v.2020_01_rev3 (Eng et al., 2015). The fasta database was further appended with contaminant proteins using Philosopher v4.4.0 (total 42,402 forward entries). The search settings were as follows: peptide mass tolerance: 10 ppm; isotope error: 0/1/2/3; number of enzyme termini: 1; allowed missed cleavages: 2; fragment bin tolerance: 0.02; fragment bin offset: 0; variable modifications: TMT-10plex tag +229.1629 for TMT experiments, and lysine + 8.0142, arginine + 10.0083 for all SILAC experiments; fixed modifications: cysteine + 57.0214. The search results were further reranked and filtered using Percolator v3.0 (The et al., 2016) at a 5% FDR. Following database search, the mzML files and Percolator PSMs were input to the SPLAT pipeline. The dynamic SILAC data were analyzed using RIANA v0.7.1 (Hammond et al., 2022) to integrate the peak intensity within a 25 ppm error of the light (+0), heavy (+8, +10), and double K/R (+16, +18, +20) peptide peaks over a 20 second retention time window encompassing the first and last MS2 scan where the peptide is confidently identified. We then calculated the fractional synthesis of all K/R containing peptides as the intensity of the 0th isotopomer peak (m0) over the sum of applicable light and heavy isotopomers (e.g., m0/m0+m8 for a peptide with one lysine). RIANA then performs intensity-weighted least-square curve-fitting using the scipy optimize function to a first-order exponential rise model to find the best-fit peptide turnover rate. Protein turnover rates are calculated as the harmonic mean of peptide turnover rates