Hypersil gold c18 column

All of the assays and controls were analyzed as described (30 (link), 40 (link)) with the following modifications for the assays that contained both prereduced wildtype (WT) PapB and QueE. A 50-μl aliquot was injected onto a Hypersil GOLD C18 column (2.1 mm x 150 mm, 1.9 mm particle size) (Thermo Fisher) preequilibrated in 0.1% (v/v) Optima TFA (Fisher) in LC-MS Optima water (Fisher). The separation consisted of washing with 100% A (0.1% [v/v] TFA in Optima water) from 0 to 3 min, followed by a linear gradient from 0 to 40% B (0.1% [v/v] TFA in Optima acetonitrile) from 3 to 20 min, followed by a linear gradient from 40% B to 100% B from 20 to 23 min, washing with 100% B from 23 to 26.5 min, and re-equilibration into 100% A from 26.5 to 30.1 min.
The assays that contained a PapB variant and QueE were analyzed as follows. A 20-μl aliquot was injected onto a Hypersil GOLD C18 column (2.1 mm x 150 mm, 1.9 mm particle size) (Thermo Fisher) preequilibrated in 0.1% (v/v) Optima TFA (Fisher) in LC-MS Optima water (Fisher). The separation consisted of washing with 100% A (0.1% [v/v] TFA in Optima water) from 0 to 3 min, followed by a linear gradient from 0 to 20% B (0.1% [v/v] TFA in Optima acetonitrile) from 3 to 20 min, followed by a linear gradient from 20% B to 100% B from 20 to 23 min, washing with 100% B from 23 to 26.5 min, and re-equilibration into 100% A from 26.5 to 30.1 min.

¹H-, ¹³C- and ¹⁹F-NMR spectra were recorded on DRX500 or ARX-800 spectrometers (Bruker, Billerica, MA, USA) in [D₆] DMSO or CDCl₃ with or without the internal standard of TMS at 0.05 or 0.1% v/v. The purity of all final compounds was > 95% purity as assessed by HPLC. Final compounds were analyzed on a 1200 series chromatograph (Agilent, Santa Clara, CA, USA). The chromatographic methods used were either: (A) Hypersil GOLD C18 column (Thermo Scientific, Waltham, MA, USA) 3 µM particle size, 150 mm length, 4.6 mm ID) or (B) a Thermo Scientific Hypersil GOLD C18 column (3 µM particle size, 250 mm length, 4.6 mm ID). UV detection wavelength = 254 nm; flow rate = 1.0 mL min⁻¹; solvent = acetonitrile/water. Both organic and aqueous mobile phase contain 0.1% v/v trifluoroacetic acid. The mass spectrometer used is a CMS-L Compact Mass Spectrometer (Advion, Ithaca, NY, USA) with an ESI or an APCI ionization source. Samples are submitted for analysis using either the atmospheric solids analysis probe (ASAP) or flow injection analysis (FIA). Compounds were prepared according to the following protocols and are detailed below. Intermediates 13a (Catalog #V4659, AK Scientific, Union City, CA, USA) and 13b (Catalog #7975AH, AK Scientific) were purchased from commercial vendors. These intermediates were left in Scheme 3 for continuity.

The PAs in the samples were measured using a LC-MS/MS system consisting of an UHPLC (Ultimate 3000, Thermo Scientific, San Jose, CA, USA) and a Triple Stage Quadrupole mass spectrometer (TSQ Vantage, Thermo Scientific, San Jose, CA, USA), as described previously with minor modifications [21 (link)]. Briefly, chromatographic separation was achieved on 150 × 2.1 mm, 1.9 μm particle sizes, C18 Hypersil Gold column fitted with a guard column (Thermo Scientific, Dreieich, Germany). Eluent A was 100% water with 0.1% formic acid and 5 mM of ammonium formate. Eluent B was 95% methanol and 5% water with 0.1% formic acid and 5 mM of ammonium formate. A stepwise gradient elution was conducted as follows: 0–0.5 min for 95% A/5% B, 7.0 min for 50% A/50% B, 7.5 min for 20% A/80% B, 7.6–9.0 min for 100% B and 9.1–15 min for 95% A/ 5% B. A flow rate of 300 μL/minute was applied and 10 μL of each sample was injected. The column temperature was maintained at 40 °C. Details of the mass parameters are listed in supporting data S1, Table S1.