LC-MS/MS for Identifications

Aqueous samples were resuspended in optima grade water with 0.1% FA at 10 mg/mL. LC-MS/MS was performed with a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Waltham, MA, United States) equipped with a Kintex C18 column (2.1 mm internal diameter × 150 mm length, 1.7 μm particle size; Phenomenex, Torrance, CA, United States) with a corresponding guard column, and a Q Exactive MS (Thermo Fisher Scientific, Waltham, MA, United States). For separation, the column temperature was 35°C, and the mobile phases were optima grade water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). A 35-min gradient at a flow rate of 0.3 mL/min with the following conditions was used: 0–5 min, held at 1% B; 5–10 min, linear gradient from 1–3% B; 10–18 min, linear gradient from 3–40% B; 18–22 min, linear gradient from 40–80% B; 22–27 min, column cleaning at 95% B; and 27–35 min, re-equilibration at 1% B. The injection volume was 4 μL, and the samples were kept at 10°C during analysis. The MS was operated in the positive ion mode with a scan range of m/z 100–1500 using a top five method for MS/MS. A target list, which included m/z more prevalent in either the control nodules or salt nodules, was used to acquire MS/MS on target m/z. If less than 5 m/z on the target list were found, then the most abundant m/z were chosen. The MS parameters were as follows: 70,000 resolution, 1E6 AGC, and 100 ms maximum injection time. The settings for HCD MS/MS were as follows: 35,000 resolution, 1E5 AGC, 100 ms max inject time, 15 s dynamic exclusion, and collision energies of 30, 35, and 40 for injections 1, 2, and 3, respectively. MetFrag (Ruttkies et al., 2016) was used to analyze the MS/MS results by searching the [M+H]⁺, [M+Na]⁺, and [M+K]⁺ adducts against the KEGG database with 5 ppm error tolerance. The in silico fragmentation was matched up to the top 20–40 experimental fragments of the MS/MS spectra at a 5 ppm and 0.01 Da tolerances. MS/MS spectra from the mzCloud high-resolution MS/MS database was used where possible to validate the MetFrag identification. For mzCloud analysis, LC-MS/MS results were loaded into Compound Discoverer software (Thermo Fisher Scientific). Briefly, raw files were aligned with adaptive curve setting with 5 ppm mass and 1.0 min retention time tolerances. Unknown compounds were detected with a 5 ppm mass tolerance, three signal to noise ratio, and 1,000,000 minimum peak intensity, and then grouped with 5 ppm mass and 0.1 min retention time tolerances. A search against the mzCloud database was then performed against all activation types with a 25 activation energy tolerance, and the intensity threshold set to true. Identifications were made if the top result in MetFrag explained almost all the major fragments and there were no other strong results in the lower scoring MetFrag results (score less than 0.8 for all other hits) or if the top result in MetFrag explained almost all the major fragments, and the compound discoverer MS/MS for this compound matched almost exactly. Arginine, soyasaponin I, asparagine, and adenosine standards were obtained to verify identifications. MS/MS parameters were the same as described for the extractions.