Optical rotations were measured on an A. Krüss Optronic P8000 polarimeter. The IR spectra were collected on a Perkin Elmer Spectrum One FTIR spectrometer. The HR-ESIMS spectra were recorded on a Waters LCT Premier XE mass spectrometer. The ESIMS spectra were recorded on an Agilent 1260 LC system equipped with a DAD detector and coupled to an Agilent 6130 Quadrupole MS with an ESI source. The NMR spectra were recorded on Bruker Avance III HD 500 or AV600 spectrometers. The ECD spectra were recorded on a Jasco J-810 spectropolarimeter with MeOH as solvent. Flash cartridge (Reveleris, HP-silica, 12 g, 20 µm), Kinetex C18 (Phenomenex, 2.6 µm, 2.1 × 100 mm), and semi-preparative C18 (Grace, 5 µm, 10 × 250 mm) were used. All solvents used for extraction were analytical grade, and solvents for HPLC were HPLC grade.
1260 lc system
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 1260 LC system is a high-performance liquid chromatography (HPLC) instrument designed for analytical applications. It is capable of performing separations, purifications, and quantitative analysis of complex mixtures. The 1260 LC system provides reliable and accurate results across a wide range of sample types and applications.
Automatically generated - may contain errors
Lab products found in correlation
Av 600, by Bruker (1 mentions)
6130 quadrupole ms, by Agilent Technologies (1 mentions)
Avance 3 hd 500, by Bruker (1 mentions)
Kinetex c18, by Phenomenex (1 mentions)
J 810, by Jasco (1 mentions)
6530 accurate mass q tof lc ms, by Agilent Technologies (1 mentions)
Extend c18 rp uplc column, by Agilent Technologies (1 mentions)
Masshunter, by Agilent Technologies (1 mentions)
Chemstation, by Agilent Technologies (1 mentions)
Zorbax sb c18 column, by Agilent Technologies (1 mentions)
Maldi tof mass spectrometry, by Bruker (1 mentions)
600 mhz nmr spectrometer, by Bruker (1 mentions)
6530 accurate mass q tof mass spectrometer, by Agilent Technologies (1 mentions)
Du800 spectrophotometer, by Beckman Coulter (1 mentions)
J 810 spectropolarimeter, by Jasco (1 mentions)
Ft ir 4100 spectrometer, by Jasco (1 mentions)
P 2000 polarimeter, by Jasco (1 mentions)
Lc ms system, by Agilent Technologies (1 mentions)
Jetstream source, by Agilent Technologies (1 mentions)
6460 qqq mass spectrometer, by Agilent Technologies (1 mentions)
24 protocols using 1260 lc system
1
Characterization of Natural Products
2
Extracting and Analyzing Crude Extracts
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The supernatant and pellets were extracted with ethyl acetate (1:1, v/v). The extracts were dried with Na2SO4 and evaporated to dryness under reduced pressure to yield crude extracts. After being weighed, the extracts were dissolved in methanol to obtain a final concentration of 1 mg/mL. LC and LC-MS/MS analyses were carried out after filtration through 0.2-μm Acrodisc MS syringe filters (25 mm).
The samples were injected as 20 μL into an Agilent 1260 LC system with an Agilent Extend-C18 RP UPLC column (2.1 × 100 mm, 1.8 μm) connected to an Agilent 6530 Accurate-Mass Q-TOF LC/MS. The LC gradient was as follows: 10% (v/v) acetonitrile (ACN) (0.1% water, 0–3 min), 10–100% (v/v) ACN (0.1% water)/0.1% water (3–23 min), 100% ACN (0.1% water, 23–25 min), 10% (v/v ACN (0.1% water, 25–30 min). The column compartment temperature was 25 °C.
Q-TOF MS settings during the LC gradient were as follows: acquisition mass range m/z 100–1600, MS scan rate 1s-1, MS/MS scan rate 2s-1, fixed collision energy 20 eV, source-gas temperature 300 °C, gas flow 11 L min-1, nebulizer 45 psi, ion polarity positive; scan source parameters—VCap 3000, Fragmentor 100, Skimmer1 65, OctopoleRFPeak 750. The MS was autotuned using Agilent tuning solution in positive mode before each measurement. LC (DAD) and MS data were analyzed with ChemStation and MassHunter software (Agilent), respectively.
The samples were injected as 20 μL into an Agilent 1260 LC system with an Agilent Extend-C18 RP UPLC column (2.1 × 100 mm, 1.8 μm) connected to an Agilent 6530 Accurate-Mass Q-TOF LC/MS. The LC gradient was as follows: 10% (v/v) acetonitrile (ACN) (0.1% water, 0–3 min), 10–100% (v/v) ACN (0.1% water)/0.1% water (3–23 min), 100% ACN (0.1% water, 23–25 min), 10% (v/v ACN (0.1% water, 25–30 min). The column compartment temperature was 25 °C.
Q-TOF MS settings during the LC gradient were as follows: acquisition mass range m/z 100–1600, MS scan rate 1s-1, MS/MS scan rate 2s-1, fixed collision energy 20 eV, source-gas temperature 300 °C, gas flow 11 L min-1, nebulizer 45 psi, ion polarity positive; scan source parameters—VCap 3000, Fragmentor 100, Skimmer1 65, OctopoleRFPeak 750. The MS was autotuned using Agilent tuning solution in positive mode before each measurement. LC (DAD) and MS data were analyzed with ChemStation and MassHunter software (Agilent), respectively.
3
Peptide Purification and Characterization
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After synthesis, peptide was purified
using an Agilent 1260 LC system equipped with an Agilent ZORBAX SB-C18
column (9.4 × 250 mm2), 5 μm particle size employing
water (A) and acetonitrile (B) mobile phase with 0.1% TFA. Crude peptide
was eluted using the following method with a flow rate of 3.0 mL/min:
gradient from 5% B to 45% B over 20 min. Absorbance at 215 and 280
nm was used to observe desired peptide peaks, and these peaks were
eluted and collected using an automated fraction collector. The purity
of the collected fractions was determined using matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry
(Bruker). Fractions deemed pure were combined and then lyophilized.
Peptide stock solutions were prepared at 20 mM concentrations in DMF.
using an Agilent 1260 LC system equipped with an Agilent ZORBAX SB-C18
column (9.4 × 250 mm2), 5 μm particle size employing
water (A) and acetonitrile (B) mobile phase with 0.1% TFA. Crude peptide
was eluted using the following method with a flow rate of 3.0 mL/min:
gradient from 5% B to 45% B over 20 min. Absorbance at 215 and 280
nm was used to observe desired peptide peaks, and these peaks were
eluted and collected using an automated fraction collector. The purity
of the collected fractions was determined using matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry
(Bruker). Fractions deemed pure were combined and then lyophilized.
Peptide stock solutions were prepared at 20 mM concentrations in DMF.
4
Comprehensive Analytical Characterization
5
LC-MS Metabolomics Profiling Protocol
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LC-MS was performed on an Agilent LC/MS system consisting of an Agilent 1260-LC system equipped with a single quadruple mass detector and electrospray ionization (ESI) interface (Agilent Technologies, Santa Clara, CA, USA). The column and mobile phase composition are the same as in the HPLC analysis, except that formic acid was used instead of methanesulfonic acid. The LC gradient program was set as follows: Time (min)/% of solvent B: 0/5, 40/15, 50/50, 55/95, 60/95, 60.1/5, 65/5. The flow rate was 1.0 mL/min for a total run time of 65 min. The mass instrument was operated in positive-ion ESI mode. Optimized mass conditions are as follows: drying gas (N2) flow rate of 12.0 L/min, drying gas temperature 300 °C, nebulizer pressure 50 psig, capillary voltage 3.0 kV. Scans were acquired from 50 to 800 amu with a 0.1 s/scan. The high-resolution mass spectra and MS/MS were recorded on a Q-TOF micro YA019 instrument (Waters, Milford, MA, USA).
6
Quantitative Proteomic Analysis by LC-MS/MS
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An Agilent 1260 LC system operating in normal flow mode at 400 μl min−1 was coupled to an Agilent 6460QQQ Mass Spectrometer equipped with an Agilent Jet Stream source and running MassHunter version B.05.00; 10 μg of peptides was separated on an Ascentis Express Peptide C18 column [2.7 μm particle size, 160 Å pore size, 5 cm length × 2.1 mm i.d., coupled to a 5 mm × 2.1 mm i.d. guard column with similar particle and pore size, operating (CG1) at 60°C; Sigma‐Aldrich]. Peptides were ionized by using an Agilent Jet Stream source operating in positive‐ion mode with the following parameter settings: sheath gas flow = 11 l min−1; sheath gas temperature = 400°C; nozzle voltage = 1000 V; nebulizing pressure = 45 psi; chamber voltage = 5000 V. A 25‐min method with the following gradient was used: 95% Buffer A (2% acetonitrile, 0.1% formic acid), 5% Buffer B (98% acetonitrile, 0.1% formic acid). Buffer B is increased to 40% over 17 min, followed by an increase to 80% B in 30 sec, where it is held for 1 min. Buffer B is ramped back down to 5% in 30 sec and equilibrated for 6 min prior to the next injection. Peptide quantification was achieved by summing the integrated peak areas of two validated SRMs. Summed peaks were averaged for all peptides associated with subcellular compartments.
7
Seaweed Arsenic Speciation Analysis
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We weighed approximately 100 mg of dried seaweed into 15 ml centrifuge tubes (VWR trace metal clean) and added 10 ml of 2% HNO3 to the tubes. The samples were then extracted using a MARS6 microwave digestion unit (CEM, Matthews, NC) with a 15-min ramp to 80 °C and a hold time of 45 min. One reference material (NIST Kelp 3232), one sample duplicate, and one sample spike per 20 samples were included in each extraction batch. Prior to analysis we added H2O2 (1% v/v) to the samples to convert arsenite to arsenate, which simplifies the chromatography. An Agilent 1260 LC system was interfaced to the 8900 ICP-MS for analysis by anion chromatography. A 250 mm × 2 mm Thermo Dionex column was used with an ammonium carbonate eluant at a flow rate of 0.35 ml/min. We obtained arsenic speciation standards from Spex Certiprep.
8
Characterization of Organic Compounds
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All chemicals used were of reagent grade or purchased as high-grade commercial products and used without further purification. Solvents used were purified via standard methods. 1H NMR spectra were recorded on a Bruker AVANCE III 400 MHz spectrometer. Mass spectrometric data were performed on an LTQ Orbitrap XL spectrometer. The high-performance liquid chromatography (HPLC) experiment was recorded on an Agilent 1260 LC system (USA). Fluorescent spectra were achieved with an F7000 fluorescence spectrophotometer. The UV–vis spectra were recorded on a TU 1900 UV–vis spectrometer. The Dulbecco’s modified Eagle’s medium (DMEM) that was added with 10% fetal bovine serum (FBS), was used to culture all the cell lines. The OD values of the CCK8 assay were measured by BIO-RAD xMark microplate spectrophotometer. Confocal fluorescence imaging was performed on an OLYMPUS FV1000 confocal microscopy.
9
LC-ELSD Compound Identification Protocol
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Liquid chromatogram separation was performed on an Agilent 1260 LC system. The chromatographic separation condition was the same as that used in the qualitative analysis method by LC-QTOF-MS/MS. The optimum parameters of ELSD for evaporator temperature, drift tube temperature, and carrier gas (high purity nitrogen) flow rate were 70 °C, 60 °C, and 1.6 L/min, respectively. In order to eliminate the difference in retention time between LC-MS and LC-ELSD and facilitate the comparison and identification of compounds in the LC-ELSD chromatogram, aleuritic acid was chosen as the standard to correct the LC-ELSD chromatogram; it is referred to as ion chromatogram of LC-MS. All the data obtained by the LC-ELSD method was analyzed by SEMICA 14.0 software, Metabo Analyst data analysis website (https://www.metaboanalyst.ca/
10
Quantitative Glycation Analysis of Proteins
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Reversed-phase
liquid chromatography and mass spectrometry were conducted using an
Agilent 1260 LC system connected in line to an Agilent 6530 Accurate
Mass Q-TOF employing a mobile phase of water (A) and acetonitrile
(B) with 0.1% formic acid.
Protein glycation reactions using
intact proteins were injected onto a Zorbax RRHD 300 Å StableBond
C8 (2.1 × 100 mm2, 1.8 μm, Agilent) column with
the following method with a flow rate of 0.4 mL/min: isocratic at
5% B for 1.75 min, a gradient change from 5% B to 80% B over 24.25
min, gradient change from 80% B to 100% B over 0.5 min, isocratic
column washing at 100% B for 7.50 min, and reequilibration at 5% B
for 7 min. Intact protein deconvolutions were generated using Agilent
MassHunter BioConfirm Qualitative Analysis software.
Peptide
glycation reactions were injected onto an AdvanceBio Peptide
2.7 μm column (2.1 × 150 mm2, Agilent) with
the following method with a flow rate of 0.4 mL/min: isocratic at
5% B for 1.75 min, gradient change from 5% B to 40% B over 14.25 min,
gradient change from 40% B to 100% B over 4 min, isocratic column
washing at 100% B for 3 min, and reequilibration at 5% B for 7 min.
Peptide data were quantified using peak volumes determined by Agilent
MassHunter Qualitative Analysis and the MassHunter Molecular Feature
Extractor.
Percent glycation was determined by the formula below.
liquid chromatography and mass spectrometry were conducted using an
Agilent 1260 LC system connected in line to an Agilent 6530 Accurate
Mass Q-TOF employing a mobile phase of water (A) and acetonitrile
(B) with 0.1% formic acid.
Protein glycation reactions using
intact proteins were injected onto a Zorbax RRHD 300 Å StableBond
C8 (2.1 × 100 mm2, 1.8 μm, Agilent) column with
the following method with a flow rate of 0.4 mL/min: isocratic at
5% B for 1.75 min, a gradient change from 5% B to 80% B over 24.25
min, gradient change from 80% B to 100% B over 0.5 min, isocratic
column washing at 100% B for 7.50 min, and reequilibration at 5% B
for 7 min. Intact protein deconvolutions were generated using Agilent
MassHunter BioConfirm Qualitative Analysis software.
Peptide
glycation reactions were injected onto an AdvanceBio Peptide
2.7 μm column (2.1 × 150 mm2, Agilent) with
the following method with a flow rate of 0.4 mL/min: isocratic at
5% B for 1.75 min, gradient change from 5% B to 40% B over 14.25 min,
gradient change from 40% B to 100% B over 4 min, isocratic column
washing at 100% B for 3 min, and reequilibration at 5% B for 7 min.
Peptide data were quantified using peak volumes determined by Agilent
MassHunter Qualitative Analysis and the MassHunter Molecular Feature
Extractor.
Percent glycation was determined by the formula below.
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