Agilent 1290 infinity lc

To perform quantitative analysis, ultra-high performance liquid chromatography triple quadrupole mass spectrometry analyses of aqueous extracts, including carnitine, acetyl-carnitine, and ocatonyl-carnitine, were performed on an Agilent 1290 Infinity LC and an Agilent 6495 Triple Quadrupole MS system equipped with an Agilent jet stream ESI source (Agilent Technologies, Palo Alto, CA, USA). Separations and the binary gradient system were identical to the UPLC/Q-TOF-MS analysis of the aqueous extracts. The column effluent was introduced into a triple quadrupole mass detector operating in a positive or negative ESI mode. Samples were analysed via the simple reaction monitoring mode for transition of the parent ion to the product ion. Characteristics of multiple reaction monitoring (MRM), calibration curve, and accuracy are shown in Supplementary Table 1.

The assay was performed on LC-Q/TOF-MS using Agilent 1290 Infinity LC coupled with an electrospray ionization (ESI) source and Agilent 6530Q-TOF mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). The LC separation was carried out on a ZORBAX RRHD SB-C₁₈ column (2.1 × 100 mm, 1.8 μm, Agilent) with the column temperature maintained at 40°C. The mobile phases consisted of ultrapure water (A) and acetonitrile (B) both containing 0.1% (v/v) formic acid; the gradient program was shown in Table 1. The injected volume of sample was 4 μL and temperature of autosampler was 4°C.
Nitrogen was used as both nebuliser gas and cone gas. Detection mode of flight tube was type V. The mass spectrometric data was collected in both positive and negative modes with the following parameters: capillary voltage 4 kV (positive mode) and 3.5 kV (negative mode), sampling cone voltage 35 kV (positive mode) and 50 kV (negative mode), source temperature 100°C, desolvation temperature 350°C (positive mode) and 300°C (negative mode), cone gas flow rate 50 L/h, desolvation gas flow rate 600 L/h (positive mode) and 700 L/h (negative mode), extraction cone voltage 4 V, and full scan mode scanning from m/z 50–1000 with a scan time of 0.03 s and an interscan time of 0.02 s. Leucine enkephalin was used as the lock mass ([M+H]⁺ = 556.2771 in the positive mode and [M−H]⁻ = 554.2615 in the negative mode).

After three days of light treatment, 8 g of soybean hypocotyls were taken and divided into 8 portions, washed with PBS, wrapped in tinfoil, frozen in liquid nitrogen, and then stored at −80 °C for subsequent metabolomic analysis. For metabolomic analysis, eight replicates were set, and the method of Wang et al. [57 (link)] was used. First, the hypocotyl samples were dissolved in 1 mL precooled methanol/acetonitrile/water solution (2:2:1, v/v/v). After vortexing, low-temperature ultrasound, centrifugation, and vacuum drying, 100 μL acetonitrile aqueous solution (acetonitrile: water = 1:1, v/v) was redissolved for mass spectrometry analysis. The supernatant was analyzed after vortexing and centrifugation. Next, an Agilent 1290 Infinity LC ultrahigh-performance liquid chromatography (UHPLC) HILIC column was used for separation, and an AB Triple TOF 6600 mass spectrometer was used to collect the primary and secondary spectra of the samples. The parameters were as described by Wang et al. [57 (link)]. In order to minimize the effect of instrument detection signal fluctuations, samples were analyzed continuously and based on a random process. To monitor and evaluate the stability of the system and the reliability of the experimental data, QC samples were added to the sample queue.

Approximately 1 × 10⁷ imDCs, mDCs and Met-mDCs (six duplicate samples of each group) were harvested and washed twice with 0.9% NaCl. The cell pellets were then quickly frozen with liquid nitrogen and transported on dry ice to Shanghai Applied Protein Technology Co., Ltd. Untargeted metabolomics profiling of the cells was performed using ultrahigh-performance liquid chromatography (Agilent 1290 Infinity LC) coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS). The number of the untargeted metabolomics profile is P20210200970.

Quantifications of phytohormone levels were conducted according to a method described previously (Wu et al., 2007 (link)). In brief, at 2 d and 4 d post infiltration, approximately 500 mg infiltrated leaves from 10 individual plants with the same treatment were harvested and pooled to generate one biological replicate. Samples were homogenized in 1 mL acetonitrile (1% famic acid). Four nanogram of D4-SA, D6-JA, 1 ng JA-Ile-D6 to were added to the aqueous extraction solvent as internal standards. All samples were then vortexed for 2 min and centrifuged at 14,000 rpm for 10 min at 4°C. Eight hundred microlitres of supernatants was collected and evaporated to dryness using a vacuum concentrator to dry. Residues were resuspended in 200 µL of acetonitrile: H₂O (1:1, v/v) and centrifuged at 14,000 g for 10 min. The supernatants were then collected and analyzed with a high-performance liquid chromatography-tandem mass spectrometry system (Agilent 1290 Infinity LC, QTRAP 5500, AB SCIES). Wheat leaves incubated with the pEDV6 empty vector were used as control groups. There were five biological replicates for each treatment and time point.