Stable isotope labeling by amino acids in cell culture (SILAC) coupled to data-dependent acquisition (DDA) is a common approach to quantitative proteomics with the desirable benefit of reducing batch effects during sample processing and data acquisition. More recently, using data-independent acquisition (DIA/SWATH) to systematically measure peptides has gained popularity for its comprehensiveness, reproducibility, and accuracy of quantification. The complementary advantages of these two quantitative techniques logically suggests combining them. Here, we develop a SILAC-DIA-MS workflow using free, open-source software. We determine empirically that using DIA achieves similar peptide detection numbers as DDA and that DIA improves the quantitative accuracy and precision of SILAC by an order of magnitude. Finally, we apply SILAC-DIA-MS to determine protein turnover rates of cells treated with bortezomib, an FDA-approved proteasome inhibitor for multiple myeloma and mantle cell lymphoma, where we observe that SILAC-DIA produces more sensitive protein turnover models. Of the proteins determined differentially degraded by both acquisition methods, we find known ubiquitin-proteasome degrands such as HNRNPK, EIF3A, and IF4A1/EIF4A-1, and a slower turnover for CATD, a protein implicated in invasive breast cancer. With improved comprehensive quantification from DIA, we anticipate making SILAC-based experiments more sensitive and reproducible, especially pulse chase SILAC for protein turnover.

Orbitrap liquid chromatography-mass spectrometry for human cell line samples. Peptides were analyzed with a Thermo Dionex UPLC coupled with a Thermo Q-Exactive HFX tandem mass spectrometer. We used an in-house pulled column created from 75 μm inner diameter fused silica capillary packed with 3 μm ReproSil-Pur C18 beads (Dr. Maisch) to 30 cm. Solvent A was 0.1% formic acid in water, while solvent B was 0.1% formic acid in 80% acetonitrile. For each injection, we loaded approximately 1 μg peptides and separated them using a 90-minute gradient from 5 to 35% B, followed by a 50 min washing gradient. For data dependent acquisition (DDA) analysis, a Top20 method was used (default charge state 3, minimum AGC target 5e4, charge exclusion 1 and >8, and dynamic exclusion 15s) with full MS (resolution 120,000; AGC 1e6, maximum IT 40 ms) and data dependent-MS2 (resolution 15,000; AGC 2e5, max IT 40 ms, isolation window 2.0 m/z, NCE 27)). For data independent acquisition (DIA) analysis of the dilution series and bortezomib experiments, we performed chromatogram library experiments as described in Searle et al. Briefly, we acquired 6 chromatogram library acquisitions with 4 m/z DIA spectra (4 m/z precursor isolation windows at 30,000 resolution, AGC target 1e6, maximum inject time 60 ms, 27 NCE) using a staggered (also referred to as overlapping) window pattern from narrow mass ranges using window placements optimized by Skyline (i.e., 398.43–502.48, 498.48–602.52, 598.52–702.57, 698.57–802.61, 798.61–902.66, and 898.6–1002.70 m/z). We acquired corresponding precursor spectra matching the range (i.e., 390–510, 490–610, 590–710, 690–810, 790–910, and 890–1010 m/z) using an AGC target of 1e6 and a maximum inject time of 60 ms were interspersed every 25 MS/MS spectra. For all single-injection acquisitions, the Thermo Q-Exactive HFX was configured to acquire either 75 × 8 m/z (covering 400-1,000 m/z) precursor isolation window DIA spectra (15,000 resolution, AGC target 1e6, maximum inject time 20 ms, 27 NCE) using an optimized staggered window pattern. Precursor spectra (target range ± 15 m/z at 60,000 resolution, AGC target 1e6, maximum inject time 60 ms) were interspersed every 75 MS/MS spectra. Isolation window schemes for the pulse SILAC experiments have been previously described (Pino et al., 2020) and the additional windowing schemes benchmarked in this work including method settings are detailed in Supplemental Table 3. QqTOF Liquid-Chromatography-Mass Spectrometry Acquisitions of E. coli samples. Briefly, samples were analyzed by reverse-phase HPLC-ESI-MS/MS using an Eksigent Ultra Plus nano-LC 2D HPLC system (Dublin, CA) with a cHiPLC system (Eksigent) which was directly connected to a quadrupole time-of-flight (QqTOF) TripleTOF 6600 mass spectrometer (SCIEX, Concord, CAN). After injection, peptide mixtures were loaded onto a C18 pre-column chip (200 µm x 0.4 mm ChromXP C18-CL chip, 3 µm, 120 Å, SCIEX) and washed at 2 µl/min for 10 min with the loading solvent (H2O/0.1% formic acid) for desalting. Subsequently, peptides were transferred to the 75 µm x 15 cm ChromXP C18-CL chip, 3 µm, 120 Å, (SCIEX), and eluted at a flow rate of 300 nL/min with a 3 h gradient using aqueous and acetonitrile solvent buffers (Burdick & Jackson, Muskegon, MI). For quantification, all E. coli peptide samples were analyzed by data-independent acquisition (DIA), using 64 variable-width isolation windows (Collins et al., 2017; Schilling et al., 2017). The variable window width is adjusted according to the complexity of the typical MS1 ion current observed within a certain m/z range using a DIA ‘variable window method’ algorithm (more narrow windows were chosen in ‘busy’ m/z ranges, wide windows in m/z ranges with few eluting precursor ions). DIA acquisitions produce complex MS/MS spectra, which are a composite of all the analytes within each selected Q1 m/z window. The DIA cycle time of 3.2 sec included a 250 msec precursor ion scan followed by 45 msec accumulation time for each of the 64 variable SWATH segments.

Cell culture medium. Cell culture medium was prepared from Dulbecco’s Modified Eagle Media (DMEM) (Thermo Fisher) supplemented with 10% dialyzed fetal bovine serum (FBS) (Atlanta Biologicals) and 1% penicillin & streptomycin (Gibco, Life Technologies Corporation, Grand Island, NY, USA). For SILAC experiments, DMEM for SILAC (Thermo Fisher) which is deficient in arginine, and lysine is supplemented with 10% dialyzed fetal bovine serum (dFBS) and 1% Pen/Strep. Light SILAC media is supplemented with L-arginine HCl (84 mg/L), L-lysine HCl (146 mg/L) and L-proline (1000 mg/L). Heavy SILAC media is supplemented with with L-arginine-13C6, 15N4 HCl (88.2 mg/L), L-lysine-13C6, 15N2 HCl (190.59 mg/L) and L-proline (1000 mg/L) (Cambridge Isotope Laboratories, Andover, MA). Media components were mixed and sterile-filtered through a 0.22 um PES membrane filter (Millipore). HeLa SILAC labeling for benchmarks. HeLa cells were cultured in heavy- and light-SILAC culture media continuously for 11 days. The labeled media were removed and the cells were quickly washed twice with sterile PBS at room temperature, scraped off the dishes, pelleted by centrifugation, snap-frozen in liquid nitrogen, and stored at −80 °C. Bortezomib pulse-SILAC labeling. Primary human foreskin fibroblasts (HFF) were cultured in DMEM, 10% FBS, 1% Pen/Strep. Prior to pulse-SILAC labeling, HFF cells were conditioned to the SILAC media formulation over four cell passages. With each passage, HFF cells were cultured with an increasing amount of SILAC media which increased in increments of 25%. Passage 1: 75% DMEM, 25% SILAC DMEM; passage 2: 50% DMEM, 50% SILAC DMEM; passage 3: 25% DMEM, 75% SILAC DMEM; passage 4: 100% SILAC DMEM. After culture in light media, HFF were supplemented with DMSO or 1000 pM Bortezomib and concurrently the media was switched to SILAC DMEM. Cells were harvested together after 0, 2, 4, 8, 10, 24, 48, 72, 168, and 500 hours of labeling, washed in PBS, and stored in −80 °C until further analysis. Human cell line protein preparation. Cell pellets were resuspended in lysis buffer (8M urea, 75mM NaCl, 50mM Tris pH 8, 1mM EDTA pH 8) and sonicated 3x for 30s, resting on ice in between. Protein concentration was estimated by BCA (Pierce BCA Protein Assay Kit, Thermo Scientific, Rockford, IL, USA). Denatured proteins were reduced with 5 mM DTT, alkylated with 15 mM IAA, and digested overnight with 1:50 trypsin (Promega). Peptides were desalted using an MCX protocol (Oasis MCX cartridge 1cc/30 mg LP, Waters Corporation) and dried down by speed-vac. To generate light/heavy mixtures, light and heavy HeLa peptides were reconstituted to 1 ug/ul and the concentrations adjusted to 1:1 using TIC. Peptides were used to construct a calibration curve spanning three orders of magnitude via five serial dilutions (Supplementary Table 1). E. coli protein preparation. E. coli pellets were available that were either previously labeled with stable isotope labeled lysine residue (13C6-15N2-Lys) growing them in heavy SILAC media from Cambridge Isotopes (heavy samples), or they were grown in regular media (light samples). We then processed isolated frozen bacterial pellets. Cell pellets of the heavy and light E. coli strains were suspended in 6 mL of PBS and centrifuged at 4°C, 15,000 g for 20 min. The firm cell pellet was collected and re-suspended and denatured in a final solution of 6 M urea, 100 mM Tris, 75 mM NaCl. Protein lysates containing 1 mg of protein were reduced with 20 mM DTT in 100 mM Tris (37°C for 1 h), and subsequently alkylated with 40 mM iodoacetamide in 100 mM Tris (30 min at RT in the dark) (Sigma Aldrich, St. Louis, MO). Samples were diluted 10-fold with 100 mM Tris (pH 8.0) and incubated overnight at 37°C with sequencing grade trypsin (Promega, Madison, WI) added at a 1:50 enzyme:substrate ratio (wt/wt). Subsequently, samples were acidified with formic acid and desalted using HLB Oasis SPE cartridges (Waters, Milford, MA) (Keshishian et al., 2007). Proteolytic peptides were eluted, concentrated to near dryness by vacuum centrifugation, and re-suspended chromatographic (aqueous) buffer A. To generate light/heavy mixtures, light and heavy E. coli peptides were diluted spanning 400x range (Supplementary Table 2).