Orbitrap elite mass spectrometer

In-gel samples were digested with chymotrypsin at 25°C for 18 h. Peptides were extracted and desalted before being injected into a nano-liquid chromatography with tandem MS (LC/MS/MS) system where an Ultimate 3000 HPLC (Thermo-Dionex) was coupled to an Orbitrap Elite mass spectrometer (Thermo Scientific) via an Easy-Spray ion source (Thermo Scientific). Peptides were separated on a ES802 Easy-Spray column (75-μm inner diameter, 25 cm in length, 3-μm C18 beads; Thermo Scientific) with a 25-min linear gradient of 2–27% mobile phase B (mobile phase A: 2% acetonitrile, 0.1% formic acid; mobile phase B: 98% acetonitrile, 0.1% formic acid). The HPLC flow rate was 300 nl/min.
Thermo Scientific Orbitrap Elite mass spectrometer was operated in positive data-dependent LC-MS/MS mode. The resolution of the survey scan was set at 60k at m/z 400. The m/z range for MS scans was 300–1600. For MS/MS data acquisition, the decision-tree mode was activated, the minimum signal intensity required to trigger MS/MS scan was 1e4, the top ten most abundant ions were selected for product ion analysis, the isolation width was 1.9 m/z, and the dynamic exclusion window was 9 s.
Xcalibur RAW files were converted to peak list files in mgf format using Mascot Distiller. Database search was performed using Mascot Daemon (2.4.0) against NCBInr_Human database. Spectra of phosphopeptides were manually checked.

HEK293A cells were infected with lentivirus expressing SFB-tagged cGAS or an empty vector for 48 h, then infected with HSV-1 for 10 h (MOI = 3) or mock-infected. The cells were lysed with lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 10% glycerol, 1 mM EDTA, 1 mM EGTA) containing a complete protease inhibitor cocktail, followed by centrifugation at 20,000 × g for 10 min at 4 °C. The supernatants were subjected to immunoprecipitation using S-protein agarose (Millipore, Cat# 69704). Immunoprecipitates were separated by SDS-PAGE, and the gel was stained with Coomassie brilliant blue. The entire lane was cut into 2-mm gel slices, digested with Trypsin, and subjected to LC-MS/MS assays using an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). The mass spectrometry data were analyzed using Thermo Proteome Discovery (version 2.3), and tandem mass spectra were searched against the UniProt-Homo sapiens database.

Phosphopeptide samples were fractionated followed a described protocol^{46 (link)}. Vacuum-dried peptides were dissolved in pH 10 buffer (10 mM ammonium bicarbonate, pH 10, adjusted by NH₄OH) and subjected to pH 10 C18 reverse-phase column chromatography. Peptides were eluted with a step gradient of 150 μL each of 6, 9, 12, 15, 18, 21, 25, 30, and 35% ACN (pH 10), pooled into six pools and vacuum-dried for nano-high-performance liquid chromatography-tandem MS.
Peptides were loaded onto a 10-cm column with a 150-μM inner diameter, packed in-house with 1.9 μM C18, and subjected to nano-high-performance liquid chromatography-tandem MS analysis by using a nano-LC 1000 coupled with an Orbitrap Elite mass spectrometer (ThermoFisher Scientific). The peptides were separated with a 75-min discontinuous gradient of 2–24%, 4–24%, or 8–26% ACN/0.1% formic acid at a flow rate of 800 nL/minute. The mass spectrometer was set to data-dependent mode, the precursor MS spectrum was scanned at 375–1300 m/z with 240k resolution at 400 m/z (2 × 10⁶ AGC target), and the 25 strongest ions were fragmented via collision-induced dissociation with 35 normalized collision energy and 1 m/z isolation width and detected by using an ion trap with 30 s of dynamic exclusion time, 1 × 10⁴ AGC target, and 100 ms of maximum injection time.

Pepsin digestion and LC-MS analysis was done as described previously (30 (link), 31 (link)). Briefly, frozen samples were loaded onto a cryogenic autosampler and then were thawed for 60 s at 5 °C before being loaded onto an immobilized pepsin column (16-μl bed volume) at a flow rate of 20 μl/min to allow digestion of proteins. Pepsin-generated peptide fragments were collected on a C18 trap column (0.2 × 1 mm, Optimize Tech Inc., Oregon City, OR) for desalting, and flowed through a reverse phase C₁₈ column (0.2 × 50 mm, MAC-MOD Analytical Inc., Chadds Ford, PA) for fragment separation using a linear acetonitrile gradient (6.4–38.4%) over 30 min. The eluent was directed into the OrbiTrap Elite mass spectrometer (Thermo Fisher Scientific) for MS analysis. Peptide identification was done by using Proteome Discoverer software (Thermo Fisher Scientific) (30 (link), 31 (link)).