Lasso peptide stock solutions were diluted to 40 µM in a final volume of 50 µL, heated at 95 °C for 4 h, and cooled to rt. Samples were then analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-Q/TOF MS) using a Synapt ESI quadrupole TOF Mass Spectrometry System (Waters) equipped with an Acquity Ultra Performance Liquid Chromatography (UPLC) system (Waters). Lasso peptides were analyzed from 2 to 98% aq. MeCN (with 0.1% formic acid) over 24 min at a flow rate of 130 µL min−1. Total ion chromatograms were used to monitor retention times via Waters MassLynx MS Software.
Acquity ultra performance liquid chromatography system
Manufactured by Waters Corporation
Sourced in United States, United Kingdom
The Acquity Ultra Performance Liquid Chromatography (UPLC) system is a high-performance liquid chromatography (HPLC) instrument designed for analytical separation and detection of chemical compounds. The Acquity UPLC system utilizes sub-2-micron particle size columns to achieve rapid, high-resolution separations with increased sensitivity and throughput compared to traditional HPLC.
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Lab products found in correlation
Beh c18 column, by Waters Corporation (8 mentions)
Acquity uplc beh c18 column, by Waters Corporation (5 mentions)
Synapt esi quadrupole tof mass spectrometry system, by Waters Corporation (4 mentions)
Synapt g2 hdms quadrupole time of flight hybrid mass spectrometer, by Waters Corporation (4 mentions)
Simac p, by Waters Corporation (3 mentions)
Masslynx, by Waters Corporation (3 mentions)
Masslynx ms software, by Waters Corporation (2 mentions)
Phenol chloroform, by Merck Group (2 mentions)
Bonded phase t3 column, by Waters Corporation (2 mentions)
Avance 3 400 mhz, by Bruker (2 mentions)
Hitrap columns for ni nta affinity chromatography, by GE Healthcare (2 mentions)
Masslynx software, by Waters Corporation (2 mentions)
Triple quad 6500, by AB Sciex (2 mentions)
Xevo tq s mass spectrometer, by Waters Corporation (2 mentions)
Acquity uplc beh amide column, by Waters Corporation (2 mentions)
Acquity hss t3 c18 column, by Waters Corporation (2 mentions)
Zorbax eclipse xdb c18 column, by Agilent Technologies (2 mentions)
Compound discoverer software, by Thermo Fisher Scientific (2 mentions)
Quattro premier xe mass spectrometer, by Waters Corporation (2 mentions)
Xevo tq xs triple quadrupole mass spectrometer, by Waters Corporation (2 mentions)
111 protocols using acquity ultra performance liquid chromatography system
1
Mass Spectrometry Analysis of Lasso Peptides
2
UPLC-QTOF-MS for Metabolite Profiling
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A 5 µL aliquot of diluted urine, fecal, serum, or liver aqueous fraction samples was injected into an Acquity ultra-performance liquid chromatography (UPLC) system (Waters, Milford, MA, USA) and separated in a BEH C18 column with a gradient of mobile phase ranging from water to 95% aqueous acetonitrile consisting of 0.1% formic acid in a 10 min run. The LC eluate was directly introduced into a Xevo-G2-S QTOF mass spectrometer for the accurate mass measurement and ion counting. Capillary voltage and cone voltage for electrospray ionization (ESI) was maintained at 3 kV and 30 V for positive-mode detection, or at −3 kV and −35 V for negative-mode detection, respectively. Nitrogen was used as both cone gas (50 L/h) and desolvation gas (600 L/h) and argon as collision gas. For accurate mass measurement, the mass spectrometer was calibrated with sodium formate solution (range m/z 50–1000) and monitored by the intermittent injection of the lock mass leucine encephalin ([M + H]+ = m/z 556.2771 or [M + H]− = m/z 554.2615). Mass chromatograms and mass spectral data were acquired and processed by MassLynxTM software (Waters, Milford, MA, USA) in centroided format. Additional structural information was obtained by tandem MS (MS/MS) fragmentation with collision energies ranging from 15 to 45 eV.
3
In vitro cA4 Cleavage Assay
4
Metabolomics and Lipidomics Profiling of PCT and ANT Tissues
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LC-MS based metabolomics and lipidomics analyses of PCT and ANT tissues were performed as previously described [20 (link), 21 (link)]. Briefly, the extraction of metabolites was performed using a system containing menthol, MTBE and water. ACQUITY™ Ultra Performance Liquid Chromatography (UPLC) system (Waters, Milford, MA, USA) was used for chromatographic separation, followed by global metabolomics and lipidomics profiling performed by a coupled AB Sciex tripleTOF 5600 plus mass spectrometer (Applied Biosystems Sciex, Foster City, CA, USA).
5
FFA Analysis in Serum Samples
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All of serum samples were stored at −80 °C until use. Sample preparation were performed according to a modified protocol based on Püttmann et al.33 (link). Forty FFAs were analyzed using an ACQUITY ultra-performance liquid chromatography (UPLC) system (Waters Corporation, Milford, USA) equipped with a binary solvent delivery system and an auto-sampler (Waters Corporation, Milford, USA), coupled to a tandem quadrupole-time-of-flight (Q-TOF) mass spectrometry (Waters Corporation, Milford, USA)16 (link). For details, see the Supplementary information . A mixture of all the reference standards at an appropriate concentration was prepared and run after every ten serum samples for quality control. Twenty-four FFAs ratios were calculated by the absolute concentrations of product- to-precursor.
6
Melanin Content Detection by LC-MS/MS
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The content and component of melanin were detected by LC-MS/MS according to previous description [40 (link)] with some modification. The chromatographic separation was performed using an Acquity ultraperformance liquid chromatography (UPLC) system (Waters, Milford, MA, USA) consisting of a Waters ACQUITY UPLC HSS T3 (2.1 × 50 mm, 1.7 μm particle size). The mobile phase A and B was 0.1% (v/v) of formic acid in deionized water and 0.1% (v/v) of formic acid in methanol, respectively. The ratio of mobile phase A in total mobile phase was gradually decreased from 90% to 0% within 5 min. The cycle time was 5 min per injection. Analyses were performed at 40 °C at a flow rate of 0.3 mL/min. MS/MS detection was performed using a Xevo TQ triple quadrupole mass spectrometer operated in positive electrospray ionization (ESI) mode similar to Yu et al. [3 (link)]. The source temperature and desolvation temperature were 150 °C and 550 °C, respectively. The cone gas flow, desolvation gas flow and collision gas flow were 50 L/h, 1100 L/h and 0.14 mL/min (argon), respectively. The analytes were monitored in multireaction monitoring mode (MRM). Specific parameters were given as Table 2 .
7
Metabolomic Analysis of Lung Tissues
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Lung tissues were homogenized in 50% methanol containing internal standards and then centrifuged at 13,000 rpm for 10 min. An equal volume of chilled acetonitrile was added to each sample tube, and then was vortexed and incubated overnight at -20°C. Next, the tubes were again centrifuged at 13,000 rpm for 10 min at room temperature and the supernatant was transferred to fresh tubes and dried under vacuum. The dried metabolite mixture was resuspended in 100 μl of 50% methanol for mass spectrometry analysis.
Samples were injected onto a reverse-phase column using an Acquity ultra-performance liquid chromatography (UPLC) system (Waters Corporation, USA). Mass spectrometry was performed by using a Quadrupole-time-of-flight mass spectrometer operating in either negative or positive electrospray ionization mode with a capillary voltage of 3.2 kV and a sampling cone voltage of 35 V. Data were acquired in centroid mode with a mass range from 50 to 850 m/z for TOF-MS scanning.
Samples were injected onto a reverse-phase column using an Acquity ultra-performance liquid chromatography (UPLC) system (Waters Corporation, USA). Mass spectrometry was performed by using a Quadrupole-time-of-flight mass spectrometer operating in either negative or positive electrospray ionization mode with a capillary voltage of 3.2 kV and a sampling cone voltage of 35 V. Data were acquired in centroid mode with a mass range from 50 to 850 m/z for TOF-MS scanning.
8
Brain Microdialysis for Hippocampal Drug Monitoring
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A probe with a dialyzing membrane of 2 mm was used for brain sampling. The rats were anesthetized with an intraperitoneal dose of 20% (g/mL) urethane (7 mL/kg) and then mounted on a stereotactic frame. An incision was made in the scalp, and a small hole was drilled for implanting the brain microdialysis probe. A guide cannula was implanted into the hippocampus according to the Paxinos and Watson atlas22 (coordinates: 5.1 mm posterior to bregma, 5.0 mm lateral to midline, and 7.0 mm lower to tip). A microdialysis probe was slowly implanted via the guiding cannula into the hippocampus of the rat. The brain microdialysis probe was then flushed with ACSF solution at a flow rate of 2.0 μL/min and continued for the duration of the experiment. Brain samples were drawn every 10 minutes after administration of LTG, until 8 hours. The midpoints of the sampling times were used for hippocampus LTG concentration–time profiles.
Hippocampus dialysate analysis was then performed on an ACQUITY Ultra Performance Liquid Chromatography (UPLC) System (Waters Corporation, Milford, MA, USA). Separation was carried out at 40°C on an ACQUITY UPLC C18 column (100×2.1 mm internal diameter, 1.8 μm; Waters Corporation) with gradient elution water (containing 5 mM ammonium acetate) and acetonitrile as the mobile phase. The flow rate was set at 0.3 μL/min. The injection volume was 2 μL.
Hippocampus dialysate analysis was then performed on an ACQUITY Ultra Performance Liquid Chromatography (UPLC) System (Waters Corporation, Milford, MA, USA). Separation was carried out at 40°C on an ACQUITY UPLC C18 column (100×2.1 mm internal diameter, 1.8 μm; Waters Corporation) with gradient elution water (containing 5 mM ammonium acetate) and acetonitrile as the mobile phase. The flow rate was set at 0.3 μL/min. The injection volume was 2 μL.
9
Metabolite Extraction and Mass Spectrometry Analysis
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RV tissues were homogenized in 50% methanol containing internal standards and then centrifuged at 13,000 rpm for 10 min. An equal volume of chilled acetonitrile was added to each sample tube, vortexed and incubated overnight at -20°C. Tubes were centrifuged at 13,000 rpm for 10 min at room temperature; and the supernatant was transferred to fresh tubes and dried under vacuum. The dried metabolite mixture was resuspended in 100 uL of 50% methanol for mass spectrometry analysis.
Samples were injected onto a reverse-phase column using an Acquity ultra-performance liquid chromatography (UPLC) system (Waters Corporation, USA). Mass spectrometry was performed using a Quadrupole-time-of-flight mass spectrometer operating in either negative or positive electrospray ionization mode with a capillary voltage of 3.2 kV and a sampling cone voltage of 35 V. Data were acquired in centroid mode with a mass range from 50 to 850 m/z for TOF-MS scanning. Duplicates (technical replicates) of each sample were tested in positive and negative ionization modes to determine the chromatographic reproducibility.
Samples were injected onto a reverse-phase column using an Acquity ultra-performance liquid chromatography (UPLC) system (Waters Corporation, USA). Mass spectrometry was performed using a Quadrupole-time-of-flight mass spectrometer operating in either negative or positive electrospray ionization mode with a capillary voltage of 3.2 kV and a sampling cone voltage of 35 V. Data were acquired in centroid mode with a mass range from 50 to 850 m/z for TOF-MS scanning. Duplicates (technical replicates) of each sample were tested in positive and negative ionization modes to determine the chromatographic reproducibility.
10
Phenolic Profiling of Avocado Leaf Extracts
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Phenolic compounds present in the fermented and non-fermented avocado leaf extracts were analysed using an Acquity Ultra Performance Liquid Chromatography (UPLC) system (Waters Corporation, Milford, MA, USA) coupled to an electrospray ionization (ESI) source operating in the negative mode and a mass detector time of flight (TOF) micro mass spectrometer (Waters). The compounds of interest were separated on an ACQUITY UPLC BEH Shield RP18 column (1.7 μm, 2.1 × 100 mm; Waters Corporation, Milford, MA, USA) at 40 °C using the conditions and gradient previously stated [31 (link)]. H2O acidified with 1% of acetic acid and acetonitrile were used as phase A and B, respectively. Analyses were performed in triplicate. The identification of the phenolic compounds was made according to the literature. For ensuring the mass accuracy, the tolerances chosen had a score higher than 90% and error lower than 5 ppm. To quantify the phenolic compounds identified in the avocado leaf extracts, calibration curves were used for vanillic acid (y = 8.1947x + 122.91; R² = 0.9976), chlorogenic acid (y = 85.138x + 135.16; R² = 0.9978), ferulic acid (y = 16.507x + 92.06; R² = 0.9980), quercetin (y = 112.8x + 287.12; R² = 0.9957), catechin (y = 41.108x + 335.6; R² = 0.9959) and rutin (y = 26.176x + 403.46; R² = 0.9924). The results are expressed as µg/g d.w.
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