NanoLC-MS/MS for protein identification
The extracted peptides from in-gel digestion were concentrated with a Speed-Vac concentrator. Four µL of peptide digest were injected via an auto-sampler and separated with a reversed-phase liquid chromatography (RP-LC) C18 column (100Å, 2 µm, 75 µm × 500 mm) at a flow rate of 250 nL/min using an EASY-nLCTM 1200 system. Mobile phase A was water with 0.1% formic acid (FA), and mobile phase B was 80% acetonitrile with 0.1% FA. Peptides were eluted using a linear gradient with increased concentration of mobile phase B from 0 to 2% for 2 min, 2-40% for 70 min, 40-80% for 5 min, and then 80-100% for 10 min.
Peptides were acquired with a Q ExactiveTM HF-X hybrid Quadrupole Orbitrap™ mass spectrometer (Thermo Fisher Scientific, Somerset, NJ) controlled by Xcalibur with full scan MS spectra from 300 to 2000 m/z. MS was run in data dependent mode with the parent ion being analyzed in the FTMS and the top 15 most abundant ions being selected for fragmentation and analysis. Tandem mass spectrometric data was analyzed using the Proteome Discoverer v 2.2 search algorithm (Thermo Fisher Scientific, Somerset, NJ) against the customized UniProt database (www.uniprot.org; Proteome ID UP000000625 and UP000002032) with sequences from evolved enzymes and co-factors used in the biocatalytic process combined with proteome fromEscherichia coli (E. coli ) strain K12. The MS data was also searched against the proteome of E. coli BL21-DE3 strain, from which cGAS was produced, to maximize E. coli HCP detection. The precursor mass tolerance was 15 ppm and fragment mass tolerance was 0.02 Da. Proteins were considered positively identified if two or more unique peptide sequences were identified with targeted false discovery rate for both peptide and protein at 1%.