Accq tag derivatization kit

Ten microliter of reconstituted extract was transferred to an autosampler vial and was combined with 70 μL of Borate Buffer (Waters, 186003836) from Waters AccQ-Tag Derivatization Kit (target pH: 8–10) and vortexed. Twenty microliter of AccQ-Tag Derivatization Agent (Waters, 186003836) was added, vortexed and let stand for 1 min. Samples were derivatized (55°C, 10 min) and vortexed. Derivatized samples were quantified with a Waters Acquity UPLC, Xevo-μ Tandem Mass Spectrometer and an AccQ-Tag Ultra RP Column 130 Å, 1.7 μm, 2.1 mm, 100 mm column using multiple reaction monitoring (MRM) and internal standard calibration (72 (link), 73 (link)).

Samples were prepared according to the provided instructions (Waters AccQTag derivatization kit, Manchester, UK) and were applied to ultra-performance liquid chromatography (UPLC) for separation (ACQUITY UPLC system, Waters, Manchester, UK). A mass spectrometer (Xevo TQ-XS, Waters) was used for monitoring. The respective amino acid levels were analyzed by Waters MassLynx 4.2 software and quantified by Waters TargetLynx application manager (Waters, Manchester, UK).

A Waters AccQ-Tag derivatization kit (Milford, MA, USA) was used to prepare the plasma samples. Using the ACQUITY UPLC System (Waters), the samples were applied to ultraperformance liquid chromatography (UPLC) for separation. A Xevo TQ-XS (Waters) mass spectrometer was used for monitoring. The amino acid concentrations were measured by Waters MassLynx 4.2 software and quantified by TargetLynx.

Samples were spiked with an internal standard mix and dried (Concentrator plus; Eppendorf, Hamburg, Germany) at 45 °C. The dried samples were reconstituted in 20 µL HCl (20 mmol/L) and derivatized according to the AccQ-Tag derivatization kit protocol (Waters GmbH, Eschborn, Germany) by adding 70 µL borate buffer and 20 µL ACQ-Tag reagent and incubating at 55 °C for 10 min. For data acquisition, an Agilent 1290 Infinity II ultra-performance liquid chromatography (UPLC) system coupled to a QTrap 5500 LC–MS/MS system from ABSciex (Darmstadt, Germany) was used in positive ion mode. Metabolite separation of derivatized amino acids was achieved with a flow rate of 300 µL/min at 35 °C on an Extend C18-column (150 × 2.1 mm, 1.8 µm; Agilent). Raw data extraction and peak identification was performed using the SCIEX OS (2.2) software.

Roots and shoots were dried at 80 °C to a constant weight for 24 h. The dry weights of the samples were measured. The dried roots and shoots were incinerated in a muffle furnace at 300 °C for 3 h and 575 °C for 6 h. The ashes were dissolved in 10 cm³ 5% nitric acid and diluted with 5% nitric acid accordingly. The concentrations of macroelements (Na, K, Ca, and Mg) and microelements (Cu, Fe, Mn, and Zn) in the digested solution were determined using a 4100-MP ICP-OES (Agilent, Santa Clara, CA, USA) [89 (link)]. Three biological replicates were tested.
Proline contents in fresh plant tissues were measured using the AccQ-Tag derivatization kit (Waters, Milford, MA, USA) with LC-MS, and three biological replicates were examined. In brief, 20 mg lyophilized plant tissue powder was extracted with 1 cm³ water under sonication. The extract was derivatized with the Waters AccQ-Tag amino acid derivatization kit and analyzed with LC-MS [90 (link)].
H₂O₂ contents were measured using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Thermo Scientific, Waltham, MA, USA) according to manufacturer’s instructions. Each sample was subjected to three biological replicates and two technical replicates.

Metabolite extracts were derivatized using an AccQ-Tag Derivatization Kit (Waters). A 5 μL partial loop injection was used for all analyses and chromatographic separation was performed using a Dionex Ultimate 3000 UHPLC system (Thermo Scientific) coupled to a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Scientific). An AccQ-tag Ultra C18 column (2.1 × 100 mm, 1.7 μm; Waters) at 50°C was used with mobile phase A: water with 10% Eluent A concentrate (Waters) and mobile phase B: acetonitrile with 1.3% formic acid. The linear gradient used was: 0 min, 0.1% B; 0.54 min, 9.1% B; 5.74 min 21.2% B; 7.74 min, 59.6% B; 8.04 min, 90% B; 8.05 min, 90% B; 8.64 min, 0.1% B; 9.5 min, 0.1% B. The flow rate was 0.5 mL/min and the total analyzed time was 9.5 min. The Q Exactive mass spectrometer was equipped with a HESI II probe in positive ion mode with source parameters set as follows: sheath gas flow rate, 50; auxiliary gas flow rate, 20; sweep gas flow rate, 3; spray voltage, 3.7 kV; capillary temperature, 300°C; S-lens RF level, 70 and heater temperature 250°C. Scan parameters were set as follows: in-source CID, 0.0 eV; microscans, 1; resolution, 70,000; AGC target, 3 × 10⁶ ions; maximum IT, 200 ms; scan range, 70-1050 m/z.