Acquity flr detector

Immediately before this experiment, 594 μL of a 99.4 μM solution of racemic CypL in 100 mM Tris–HCl (pH 7.5) containing 300 mM l-ascorbic acid sodium salt was mixed with 6 μL of water or a 1.2 mg mL⁻¹ solution of a recombinant CypLase in 20 mM Tris–HCl (pH 8.0). The mixtures were incubated at room temperature, and 200 μL of each mixture was collected at 2 min, 7 min, and 17 min after starting the reaction, followed by immediate filtration through an Amicon Ultra-0.5 centrifugal filter device with nominal molecular weight limit (NMWL) of 10 KDa (Millipore). Ten aliquots of the resultant filtrates were subjected to chiral HPLC analysis. Chiral HPLC analysis was performed on a Waters ACQUITY UPLC H-Class system (Waters) equipped with a CHIRALCEL OZ-RH chiral column (ϕ4.6 × 150 mm, 5 μm; Daicel Chemical Industry), a multiwavelength detector (ACQUITY UPLC PDA eλ detector; Waters), and a fluorescence detector (ACQUITY FLR detector; Waters). The HPLC conditions were as follows: mobile phase, 30% (v/v) acetonitrile in a 100 mM solution of potassium hexafluorophosphate in H₂O; flow rate, 0.8 mL min⁻¹; fluorescence detection, excitation/emission, 430/570 nm.

Samples (0.1 g) were ground in liquid nitrogen to a fine powder and extracted with 0.1 M HCl (0.1 mL) by vortexing at 4 °C for 15 min. The resulting homogenates were centrifuged for 10 min at 4 °C and 16,400 g to remove cell debris. Adenosines were derivatized with chloro-acetaldehyde as previously described [66 (link)]. The metabolites were separated by reversed-phase chromatography on an Acquity HSS T3 column (100 mm × 2.1 mm, 1.7 µm, Waters) equilibrated in buffer A (5.7 mM TBAS, 30.5 mM KH2PO4 pH 5.8) by applying the following gradient: 0.6 mL 1% B, 1.9 mL 8% B, 1.9 mL 14% B, 5.7 mL 50% B. Buffer B was a mix of 34% buffer A and 66% acetonitrile. The fluorescent 1,N⁶-etheno-derivatives of MTA, SAM, and SAH were quantified with an Acquity FLR detector (Waters, excitation: 280 nm, emission: 410 nm) connected to an H-class UPLC system. The thiols (Cys, GSH, and Hcys) were labeled with monobromobimane, and the resulting fluorescent thiol-bimane derivates were separated by reversed-phase chromatography according to (Dong et al., 2017).

To analyze the content of branched-chain α-ketoacids, 100 μl of yeast conditioned medium was mixed with 200 μl cold 1M perchloric acid. Insoluble materials were removed by centrifugation, 150 μl of the resulting supernatant were mixed with an equal volume of 25 mM o-phenylendiamine solution and derivatized by incubation at 50°C for 30 min. After centrifugation the derivatized keto-acids were separated by reversed phase chromatography on an Acquity HSS T3 column (100 mm x 2.1 mm, 1.7 μm, Waters) connected to an Acquity H-class UPLC system. Prior to separation, the column was heated to 40°C and equilibrated with 5 column volumes of solvent A (0.1% formic acid in 10% acetonitrile) at a flow rate of 0.55 ml/min. Separation of ketoacid derivates was achieved by increasing the concentration of solvent B (acetonitrile) in solvent A as follows: 2 min 2% B, 5 min 18% B, 5.2 min 22% B, 9 min 40% B, 9.1min 80% B and hold for 2 min, then return to 2% B in 2 min. The separated derivatives were detected by fluorescence (Acquity FLR detector, Waters, excitation: 350 nm, emission: 410 nm). Data acquisition and processing were performed with the Empower3 software suite (Waters).

Fluorescence analysis was carried out on an Acquity UPLC H-class with Acquity FLR detector and Waters Empower 3 Software (Waters, Milford, MA, USA). Analytical column Acquity UPLC HSST3 (particle size 1.8 μm, diameter 2.1 mm, length 100 mm) at 40 °C and a binary gradient of water (A) and acetonitrile (B) with 0.1% formic acid each (v/v) was used (0.0 min 2% B, 8.0 min 100% B, 9.0 min 100% B, 9.1 min 2% B, 10 min 2% B). Injection volume was set to 2 μL and flow rate to 0.5 mL/min. Fluorescence detection (FLD) was carried out with excitation at 327 nm and emission at 390–490 nm.

The methanolic solutions of chiral-separated (R)- and (S)-CypL were filtered through a centrifugal filter Ultrafree-MC (0.22 μm; Millipore, Billerica, MA, USA) and diluted 2 fold with methanol or mixed together in a 1:1 ratio. The methanolic extract of dried luminous ostracods was prepared as follows: ten dried luminous ostracods in a commercially available kit for observation of bioluminescence of sea-firefly (Hatenouruma, Tokyo) were homogenized in 200 μL of ice-cold methanol on ice and centrifuged at 14,000× g for 3 min at 4 °C followed by filtration through an Ultrafree-MC centrifugal filter (0.22 μm; Millipore). Ten microliter aliquots of these prepared solutions were subjected to chiral HPLC analysis. Chiral HPLC analysis was performed on a Waters ACQUITY UPLC H-Class system (Waters) equipped with a CHIRALCEL OZ-RH chiral column (ϕ4.6 × 150 mm, 5 μm; Daicel Chemical Industry), a multiwavelength detector (ACQUITY UPLC PDA eλ detector; Waters), and a fluorescence detector (ACQUITY FLR detector; Waters). The HPLC conditions were as follows: mobile phase, 30% (v/v) acetonitrile in a 100 mM solution of potassium hexafluorophosphate in H₂O; flow rate, 0.8 mL min⁻¹; fluorescence detection, excitation/emission, 430/570 nm.

Non–thiol-containing amino acids were quantified after specific labeling with the fluorescence dye AccQ-TagTM (Waters) according to the manufacturer's protocol. The resulting derivatives were separated by reversed phase chromatography on an Acquity BEH C18 column (150 mm × 2.1 mm, 1.7 μm, Waters) connected to an Acquity H-class UPLC system and quantified by fluorescence detection (Acquity FLR detector, Waters). The column was heated to 42 °C and equilibrated with 5 column volumes of buffer A (140 mM sodium acetate pH 6.3, 7 mM triethanolamine) at the flow rate of 0.45 mL min^-1. Baseline separation of amino acid derivates was achieved by increasing the concentration of acetonitrile (B) in buffer A as follows: 1 min 8% B, 7 min 9% B, 7.3 min 15% B, 12.2 min 18% B, 13.1 min 41% B, 15.1 min 80% B, hold for 2.2 min, and return to 8% B for 1.7 min. Data acquisition and processing were performed with the Empower3 software suite (Waters).