Atlantis c18 column

Nucleotides were extracted using a modified method of Liu et al. (2014). Freeze‐dried mushroom powder (1.000 g) was extracted with 20 ml of distilled water and heated to the boiling temperature for 1 min. Then, it was cooled to room temperature, centrifuged at 9,850 g for 15 min and then filtered with a 0.45‐μm polyvinylidene fluoride microfiltration membrane (Shanghai Xingya Purification Material Co.) for HPLC analysis. The mushroom soup was centrifuged at 5,000 rpm for 10 min, and the liquid supernatant was filtered as described above. The nucleotide was analyzed using a Waters Atlantis C₁₈ column (250 × 4.6 mm, 5 μm). The mobile phase was 0.01 M KH₂P0₄ buffer solution including 1.40 mM tetra‐n‐butylammonium hydrogen sulfate (A) and methanol (B), and the flow rate was 1 ml/min. All samples were detected at 254 nm using a Waters e2695 Separations Module equipped with a Waters 2489 UV/Vis Detector.

The HPLC separation was performed by an Agilent 1260 Infinity II series HPLC System that was equipped with Waters Atlantis C18 column (150 × 3.9 mm, 5 μm). The mobile phase consisted of solvent A (water containing 0.1% formic acid) and solvent B (methanol). The gradient procedure was as follows: 0–3 min 60–5% A, 3–5 min 5–5% A, 5–5.02 min 5–60% A, and 5.02–6 min 60–60% A. The flow rate was 0.5 mL/min and the injection volume was 5 μL.
An Applied Biosystems Sciex QTRAP® 4500 MS/MS spectrometer equipped with a version of 1.6 Analyst software (AB SCIEX, Massachusetts, USA) was used for the analysis. The instrument was equipped with an ESI source, and the targeted analytes were performed in positive and negative ion modes for all the targeted analytes. Compressed air was used as GS1 and GS2, and high-purity (99.99%) nitrogen was used as CUR and CAD. The operation conditions were as follows: the EP was 10.0/−10.0 V, the TEM at 500 °C, the IS 5500/–4500 V, GS1 set to 55 psi, GS2 set to 55 psi, CUR set to 35 psi, and the CXP 13.0/−15.0 V for ESI⁺/ESI⁻ mode, respectively. The dwell time for each MRM transition was 10 ms.

The identities of the four chimeric proteins of MCR-1 and -2 (TM-MCR-1, TM1-EptA, TM-MCR-2, and TM2-EptA) were examined using a Waters quadrupole time of flight (QTOF) API-US mass spectrometer (53 (link), 54 (link)). A band obtained from SDS-PAGE separation of purified protein was digested with trypsin (G-Biosciences St. Louis, MO), and the resultant peptides were analyzed with a Waters Atlantis C₁₈ column (0.03-mm particle size, 0.075 by 150 mm). Finally, the acquired data were subjected to further analyses through the Waters ProteinLynx Global Server 2.2.5, Mascot (Matrix Sciences), and BLAST against the NCBI nr database.

The identity of the recombinant FadR_she protein we produced was confirmed using A Waters Q-Tof API-US Quad-ToF mass spectrometer connected to a Waters nano Acquity UPLC) (Feng & Cronan, 2011a (link)). In brief, the protein band of interest was cut from 15% SDS-PAGE gel, de-stained and digested with Sequencing Grade Trypsin (G-Biosciences St. Louis, MO, 12.5 ng/μL in 25 mmol/L ammonium bicarbonate); Second, the resulting peptides were loaded on a Waters Atlantis C-18 column (0.03 mm particle, 0.075 mm × 150 mm), following the further cleaning treatment. The data dependent acquisition combined with ms/ms analysis was routinely performed (Feng & Cronan, 2011a (link)).

Target chemicals were analyzed using a 20A HPLC system (Shimadzu, Tokyo, Japan) coupled with a Q-Trap 5500 tandem mass spectrometer (MS/MS; Applied Biosystems, Foster City, CA, USA). Chromatographic separation of the analytes was performed on an Atlantis C18 column (1.7 μm, 3.0 × 100 mm, Waters, Dublin, Ireland), accompanied by mobile phases of water and methanol. The gradient elution conditions and the mass spectrometric information of the target chemicals are shown in Tables S1 and S2, respectively. The injection volume was 5 µL and the column temperature was set at 40 °C. The flow rate was set at 0.3 mL/min. The mass spectrometer was performed in negative electron spray ionization (ESI) mode. The source temperature was set as 550 °C and the ionization voltage was −4500 V.

A Waters Q-Tof API-US Quad-ToF mass spectrometer was applied to determine the identity of S. suis BirA (BirA_ss) protein1 (link)46 (link). The purified protein band was cut from the gel and digested with Trypsin (G-Biosciences St. Louis, MO), giving a pool of overlapping peptides loaded on a Waters Atlantis C-18 column (0.03 mm particle, 0.075 mm × 150 mm). The acquired data were subjected to the ms/ms analyses.