Method 1. Samples were diluted to 10 μM (10 μl) with 25 mM ammonium bicarbonate and were digested at 37°C overnight using sequencing-grade trypsin (Promega) at 1:50. One picomole of the resultant peptides was aliquoted for MS analysis. Nanoliquid chromatography (nLC)–MS/MS data were recorded on a Sciex 5600+ mass spectrometer equipped with the Eksigent 2D Ultra NanoLC system using a Thermo Scientific Pepmap C18 column (75 μm × 15 cm) and trap (2 cm) in trap-elute configuration. The sample was held on the trap and washed for 5 min with loading buffer A (2% CH3CN, 98% H2O, 0.05% trifluoroacetic acid). The trap was switched in line with the column, and elution was performed with a linear gradient using eluent A (2% CH3CN, 98% H2O, 0.1% FA) and increasing eluent B (98% CH3CN, 2% H2O, 0.1% FA). The gradient was 95% A/5% B (at 0 min) linear to 60% A/40% B (at 6 min), linear to 5% A/95% B (at 9 min), held at 5% A/95% B until 12 min, linear to 95% A/5% B (at 14 min), and held at 95% A/5% B for 14 to 20 min. Mass spectra were acquired in positive-ion mode in information-dependent acquisition mode by performing 150 ms of MS followed by 80 ms of MS/MS analysis of the 10 most intense peaks seen by MS. These masses were then excluded from analysis for the next 10 s. MS spectra were acquired from 400 to 1350 m/z, and MS/MS spectra were acquired from 95 to 2000 m/z. MS/MS data were extracted using the SCIEX PeakView software and analyzed using the Mascot 2.6 search engine (Matrix Science) against a sequence database containing OphA (and mutants). The data were searched with tolerances of 20 ppm for the precursor and 0.1 Da for fragment ions, trypsin as the cleavage enzyme, and three missed cleavages. A methyl modification (+14.01565 atomic mass unit monoisotopic) was added to the modification database for V, G, I, and A and used as a variable modification. The potential methylation sites were further verified by manual interpretation of subsequent analysis under the same conditions as above but utilizing product ion mode scanned specifically for the masses of interest of the C-terminal trypsin-digested peptide (and mutants) in the various methylated states. The MS spectra were used to create extracted ion chromatograms of the masses of interest ±0.05 m/z, and the peak areas were measured using the PeakView software.

Method 2. In vitro samples (10 μl) were transferred into 1.7-ml Protein LoBind tubes (Eppendorf). After denaturing for 1 hour using 6 μl of 8 M urea, a final concentration of 0.5 M ammonium bicarbonate, 2 mM TCEP (pH 8.0), and Trypsin Gold (Promega) in a molar ratio of 1:50 was added to the sample and incubated overnight at 37°C. The samples were then desalted and purified using C18 ZipTips (Millipore) and eluted with 90% acetonitrile (ACN), 0.1% FA. After drying the samples in a SpeedVac (Eppendorf), peptides were resuspended in 15 μl of 20% ACN, 0.1% FA, and transferred to glass vials for MS analysis. An injection volume of 4 μl was used for MS samples. HPLC-MS/MS data were recorded on a Thermo Scientific Fusion mass spectrometer equipped with a Dionex Ultimate 3000 UHPLC system using a nLC column (200 mm × 75 μm) packed using Vydac 5-μm particles with a 300 Å pore size (Hichrom Limited). A 75-μm emitter with a 10-μm tip was used for electrospray (New Objective). Elution was performed with a linear gradient using water with 0.1% (v/v) FA (solvent A) and ACN with 0.1% (v/v) FA (solvent B) at a flow rate of 0.3 μl/min. The column was equilibrated with 20% solvent B for 5 min, which was followed by a linear increase of solvent B to 85% over 32 min and a final elution step with 85% solvent B for 2 min. Mass spectra were acquired in positive-ion mode with the following settings: spray voltage at 2200 V and an S-lens level at 60. Full MS was done at a resolution of 60,000 [automatic gain control (AGC) target, 4 × 105; maximum ion trap (IT), 50 ms; range, 300 to 1800 m/z], and data-dependent MS/MS was performed at a resolution of 15,000 (AGC target, 5 × 105; maximum IT, 500 ms; isolation window, 2.2) using higher-energy collisional dissociation (HCD) with a stepped normalized collision energy of 14, 18, and 22. The inclusion list contained masses of the trypsinized fragments encoding the omphalotin peptide (or one of the variants) and its different methylation states. Data were processed using Thermo Fisher Xcalibur software and MaxQuant, as previously described (12).

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