Optima grade methanol

Ethyl acetate (EtOAc; J.T. Baker and Fisher Scientific) and Optima-grade methanol (MeOH; Fisher Scientific) were used. P. aeruginosa PAO1 wild type or ΔpqsA cells were grown overnight in LB (RPI) at 37°C with 200 rpm shaking. Bacterial cells were centrifuged at 5,000 rpm (Biofuge pico, Heraeus) and washed with 0.5% chelex treated CAA. New cultures were seeded in 0.5% chelex treated CAA at a starting OD₆₀₀ of 0.05 and incubated at 37°C with 200 rpm shaking. After 48 h the cultures were centrifuged at 3,800 rpm (Eppendorf Centrifuge 5810 R) for 30 min at room temperature. The supernatant was decanted and filtered using a Nalgene Rapid-flow Filter Unit (0.2 µm aPES membrane; Thermo Scientific). The cell free supernatant was acidified to pH 1.8–2 with 6 M HCl. Pyochelin and pyochelin methyl ester were extracted with 3 volumes EtOAc and evaporated to dryness in a Rotavapor (Büchi; Rotavapor R-300) or a SpeedVac vacuum concentrator (SPD131DDA SpeedVac Concentrator; Thermo Scientific). Dried samples were resuspended in MeOH and stored at – 20° C. Samples were centrifuged for 5 min at 10,000 rpm (Thermo; Sorvall ST 40R) to remove nonsoluble particulates and diluted as needed in MeOH containing 1 µM glycocholic acid.

Band excision was performed in a laminar flow hood while wearing a head covering, a face mask, and extended cuff gloves over the sleeves of a full-length lab coat. To avoid cross-contamination during band excision, lanes from dinosaur, sediment, and blank GuHCl extracts were excised separately, transferred to new sterile Petri dishes, and cut into 10 gel sections, as shown in (Figure 2B). Each section was diced into ~1 mm³ cubes using a new, fresh razor rinsed in Optima-grade methanol (Fisher Scientific), followed by rinsing with Optima-grade water (Fisher Scientific) for each individual gel section. Each diced gel section was then transferred to a new 1.5 mL tube (LoBind, Eppendorf), using tweezers rinsed in a stream of methanol followed by a stream of water before every manipulation. Excised gel bands were then destained using the Pierce Silver Stain Kit for Mass Spectrometry, following manufacturer’s protocols (all buffers made in Optima-grade water and all steps at RT). In brief, pieces were incubated in 200 μL of destain solution (twice for 15 min each, with gentle agitation), then washed in 300 μL of each of the following for 15 min with agitation: 100 mM ammonium bicarbonate in Optima-grade water (ABC); 50% 100 mM ABC, 50% Optima-grade acetonitrile (ACN); and 100% ACN. Dehydrated sections were dried to completion by speed vacuum (~10 min) and stored at −80 °C.

Optima grade formic acid and β-carotene standard (≥ 97% purity) were purchased from Sigma-Aldrich (St. Louis, MO). A lycopene standard was isolated and purified following a previously published method (Kopec et al., 2010 (link)). β-Cryptoxanthin (≥ 97% purity) was purchased from Extrasynthese (Genay, France). Lutein (≥ 95% purity), zeaxanthin (≥ 98% purity), and α-carotene (≥ 95% purity) were purchased from Cayman Chemical (Ann Arbor, MI). Optima grade methanol, HPLC grade acetone, hexane, and methyl tert-butyl ether (MTBE), and ACS grade potassium hydroxide were purchased from Fisher Scientific (Pittsburgh, PA). Double-deionized water was obtained from a Millipore Q-Plus filtration system.

Sample preparation and experimental details have been reported previously for 3-ring and 4-ring MDA ESI studies^{2 (link)} and 2-ring MDA ESI and MALDI studies^{1 (link),3 (link)}, but a few details will be mentioned here. Methylenedianiline (MDA) 3-ring and 4-ring samples were provided by Dr. Stefan Wershofen (Bayer MaterialScience AG, 47812 Uerdingen, Germany). Optima grade methanol and water with 0.1% formic acid were obtained from Fisher Scientific (Waltham, MA, USA). Tetralkylammonium salts, α-cy-ano-4-hydroxycinnamic acid (CHCA) and alkali salts were obtained from Sigma-Aldrich (St. Louis, MO, USA). Samples were dissolved at a concentration of 1 mg/mL in 9:1 methanol/water containing 0.1% formic acid (v/v). Each MDA isomer was combined in a 10:1 CHCA matrix-to-analyte ratio and two or three layers of 0.5 µL of samples were spotted on a 100-well MALDI plate.

S-(+)-Campothecin was provided by Millipore Sigma (Rockville, MD), SN-38 was provided by Selleck Chemicals (Houston, TX), and NLG207 was provided by NewLink Genetics (Ames, IA). The chemical structures of camptothecin and SN-38, analyte and internal standard respectively, are shown in Figure S1. Formic acid and sodium hydroxide were provided by Millipore Sigma (Rockville, MD). Optima-grade methanol was obtained from Fisher Scientific (Fairlawn, NJ), and a Hydro-Reverse Osmosis system (Durham, NC) linked to a Milli-Q UV Plus purifying system (Billerica, MA) was used to generate de-ionized water. Drug-free human heparinized plasma and whole blood was obtained from the NIH Clinical Center Blood Bank (Bethesda, MD).