NMR spectra were collected using either a Bruker 800 MHz, Varian 500 MHz, or Bruker 300 MHz instrument. The chemical shifts reported were referenced to the residual solvent peaks of either CDCl3, DMSO-d6, and MeOH-d4. HRMS analysis was performed using an SCIEX TripleTOF 4600 mass spectrometer with Analyst TF software. MS/MS was acquired using a SCIEX Qtrap 4500 coupled to a Shimadzu Prominence UFLC system consisting of three LC-20AD pumps, a DGU-20A degassing unit, SIL-20AC auto sampler, CTO-20AC column oven and CBM-20A communication bus module. Semi-preparative and analytical HPLC was carried out using a Dionex UltiMate 3000 HPLC system equipped with a micro vacuum degasser, an autosampler, and a diode-array detector.
Triple tof 4600 mass spectrometer
Manufactured by AB Sciex
Sourced in United States
The Triple TOF 4600 is a mass spectrometer designed for high-resolution, accurate mass analysis. It utilizes a triple quadrupole architecture with a time-of-flight (TOF) analyzer to provide precise mass measurement capability. The instrument is capable of performing MS and MS/MS analysis for a wide range of applications.
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Lab products found in correlation
Du800 spectrophotometer, by Beckman Coulter (5 mentions)
Ultimate 3000 hplc system, by Thermo Fisher Scientific (4 mentions)
P 2000 polarimeter, by Jasco (4 mentions)
500 mhz instrument, by Agilent Technologies (3 mentions)
Nanorslc system, by Thermo Fisher Scientific (2 mentions)
800 mhz nmr instrument, by Bruker (2 mentions)
Uflc system, by Shimadzu (1 mentions)
Ultimate 3000 hplc, by Thermo Fisher Scientific (1 mentions)
Qtrap 4500, by Shimadzu (1 mentions)
Analyst 1, by Thermo Fisher Scientific (1 mentions)
Poroshell 300sb c8, by Agilent Technologies (1 mentions)
Lc 30ad system, by Shimadzu (1 mentions)
2998 photodiode array detector, by Waters Corporation (1 mentions)
1290 uplc system, by AB Sciex (1 mentions)
Avance av 500 spectrometer, by Bruker (1 mentions)
Luna omega c 18 column, by Shimadzu (1 mentions)
Analyst tf 1, by AB Sciex (1 mentions)
Nexera uhplc system, by Shimadzu (1 mentions)
Agilent 1260 infinity, by Agilent Technologies (1 mentions)
Lc 20ar, by Shimadzu (1 mentions)
22 protocols using triple tof 4600 mass spectrometer
1
Spectroscopic Characterization of Compounds
2
Protein Purification and HPLC-MS Analysis
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High performance liquid chromatography analysis was carried out with an LC-30AD system (Shimadzu, Japan). The purified protein samples in PBS were held at room temperature, and the injection volume was 10 μl. The chromatography column was an Agilent Poroshell 300SB-C8 (1.0 × 75 mm, 5 μm). The flow rate was 0.3 ml/min in gradient mode with 0.1% formic acid (A) and acetonitrile (B). The equilibrium time after the run was 25 min. Gradient conditions were as follows: 0–5.0 min, 5% B; 5.0–20.0 min, 5%–80% B; 20.0–25.0, 80% B. The column temperature was maintained at 60 °C. Mass spectral analysis was performed on a Triple TOF 4600 mass spectrometer (Sciex, America) equipped with an electrospray ionization (ESI) source and operated in positive ion mode. A calibration solution was used to calibrate the instrument. The curtain gas (CUR), nebulizer gas (GS1) and turbo gas (GS2) were set at 35 psi, 55 psi and 55 psi, respectively. The electrospray voltage was 5.5 kV, and the turbo ion spray source temperature was 550 °C. Nitrogen was employed as the collision gas. The mass spectrometer operated in the TOF-MS full scan mode with a 500–4000 m/z range. Data acquisition was performed using Analyst 1.6.2 software (Applied Biosystems). The Bayesian protein reconstruction tool was used to calculate protein molecules.
3
HPLC-UV/MS Analysis of Phytochemicals
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The HPLC system consists of a Waters 2995 controller and 2998 Photodiode Array detector. The separation was performed on an Elite Kromasil C18 column (250 mm × 4.6 mm, 5 μm). The flow rate was set at 1.0 mL/min, with the column temperature set at 25°C, and detective wavelength was set at 254 and 289 nm. The acetonitrile and water containing 0.2% acetic acid were employed as mobile phases A and B, respectively. The binary gradient program was set as follows: 0–10 min, 80% of B; 10–15 min, 80 to 75% of B; 15–25 min, 75% of B; 25–50 min, 75 to 35% of B; and equilibrated for 10 min before the next injection. The injection volume was 10 μL. Data were collected and visualized by Waters Empower Chemstation Software.
The chromatographic peak purity analysis was performed on an Agilent 1290 UPLC system coupled with a SCIEX Triple TOF 4600 mass spectrometer equipped with an ESI interface. The optimized MS conditions were as follows: TOF mass range between 50 and 1700, curtain gas 35 psig, ion spray voltage floating −4500/5000 kV, and ion source temperature 500°C. The collision energy was set at 10 V to obtain more fragment information.
The chromatographic peak purity analysis was performed on an Agilent 1290 UPLC system coupled with a SCIEX Triple TOF 4600 mass spectrometer equipped with an ESI interface. The optimized MS conditions were as follows: TOF mass range between 50 and 1700, curtain gas 35 psig, ion spray voltage floating −4500/5000 kV, and ion source temperature 500°C. The collision energy was set at 10 V to obtain more fragment information.
4
High-Resolution ESI Mass Spectrometry for Chemical Analysis
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High-resolution ESI− mass spectra were obtained on an LC-Q-TOF-MS/MS system, which was composed of an Agilent 1290 Infinity HPLC system coupled with a Sciex Triple TOF 4600 mass spectrometer. The chromatographic separation was accomplished on an Agilent Extent C18 (50 × 2.1 mm, 1.8 μm) column at 30 °C. A linear gradient program was used, starting at 0 min from 10% pump B and maintaining 10% pump B over 1 min (pump A, water containing 0.1% (v/v) formic acid; pump B, acetonitrile) at a flow rate of 0.3 mL/min, at 1 min from 10% pump B to 95% pump B over 5 min, maintaining 95% pump B over 3 min, at 9 min from 95% pump B to 10% pump B over 0.1 min, and stopped at 12 min. The injection volume was 1 μL. The tandem mass spectrometric analysis was performed under the ESI− mode with the settings as follows: ISV, −5000 V; TEM, 500 °C; CUR, 35 psi; GS1, 50 psi; GS2, 50 psi; declustering potential (DP) 100 V; collision energy (CE), 5 V.
NMR data were collected on a Bruker Instruments Avance AV500 spectrometer (500 MHz) (Bruker Corporation, Billerica, MA, USA).
NMR data were collected on a Bruker Instruments Avance AV500 spectrometer (500 MHz) (Bruker Corporation, Billerica, MA, USA).
5
UHPLC-HRMS Analysis of Methanolic Extract
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The methanolic extract was investigated using the NEXERA UHPLC system (Shimadzu, Tokyo, Japan) equipped with a Luna® Omega C-18 column (50 × 2.1 mm i.d., 1.6 μm particle size). Two μL of each sample were injected. The mobile phase was constituted by water (solvent A) and acetonitrile (solvent B), both acidified with formic acid (0.1% v/v). A linear gradient was used as follows: 0–10 min, 5%→32% B; 10–28 min, 32→75% B; 28-29 min, 75%→95% B; 29–30 min, 95% B; 30–32 min, column re-equilibration. The flow rate was set at 400 μL/min. High-Resolution Mass Spectrometry (HR-MS) data were obtained by an AB SCIEX Triple TOF® 4600 mass spectrometer (AB Sciex, Concord, ON, Canada), equipped with a DuoSprayTM ion source (AB Sciex, Concord, ON, Canada) operating in the negative ElectroSpray (ESI) mode. A full-scan Time-Of-Flight (TOF) survey and 8 information-dependent acquisition MS/MS scans were acquired, using the following parameters: curtain gas 35 psi, nebulizer and heated gases 60 psi, ion spray voltage 4500 V, ion source temperature 600 °C, declustering potential −80 V and collision energy −40 ± 15 V. The instrument was controlled by Analyst® TF 1.7 software (AB Sciex, Concord, ON, Canada), whereas MS data were processed by PeakView® software version 2.2 (AB Sciex, Concord, ON, Canada).
6
Analytical Techniques for Compound Characterization
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The UV spectrum was recorded on a Jasco V-560 spectrophotometer (JASCO Corporation, Kyoto, Japan). Optical rotation was obtained on an Autopol IV Polarimeter (Rudolph Research Analytical, Flanders, NJ, USA). CD spectrum was acquired on a Jasco J-810-150S spectropolarimeter (JASCO Corporation, Japan). High-resolution electrospray ionization mass spectrometry (HRESIMS) data were collected on an AB Sciex Triple TOF 4600 mass spectrometer (AB SCIEX, Framingham, MA, USA). NMR spectra were recorded on a Bruker Avance II 500 MHz NMR spectrometer (Bruker, Karlsruhe, Germany) with tetramethylsilane (TMS) as an internal standard. Agilent 1260 Infinity (Agilent Technologies Inc., Santa Clara, CA, USA), Waters 2535 (Waters Corporation, Milford, MA, USA), and Shimadzu LC-20AR (Shimadzu Corporation, Kyoto, Japan) semi-preparative HPLC systems were created using a Welch Ultimate XB-C18 column (250 mm × 10.0 mm, 5 μm). Silica gel (100−200 mesh and 200−300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China) and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were used for column chromatography. The Silica gel GF254 (Qingdao Marine Chemical Co., Ltd., Qingdao, China) was used for analytical and preparative thin-layer chromatography (TLC).
7
Peptide Fractionation and Mass Spectrometry
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The peptide mixture was diluted 10-fold with water/formic acid (pH 3.0) and loaded onto an strong cation exchange chromatography column (SCIEX, 4326695) and fractionated into ten subgroups using isotope-coded affinity tag Cation Exchange Buffer (SCIEX, 4326747). Each strong cation exchange fraction was desalted using reverse-phase (RP) chromatography.
The fractions were separated by nano-high performance liquid chromatography (Eksigent Technologies, Dublin, CA, USA) using a secondary RP analytical column (Eksigent, C18, 3 μm, 150 mm × 75 μm). Peptides were subsequently eluted using the following gradient conditions: phase B (98% acetonitrile with 0.1% formic acid) from 5% to 45% B (5–100 minutes). The total flow rate was maintained at 300 nL/minute. An electrospray voltage of 2.3 kV vs the inlet of the mass spectrometer was used. An AB SCIEX TripleTOF™ 4600 mass spectrometer (Foster city, CA, USA) was operated in information-dependent data acquisition mode to automatically switch between MS and MS/MS acquisition. MS spectra were acquired across the mass range of 350–1,250 m/z using an accumulation time of 250 ms per spectrum. MS spectra, scanned from 100 to 1,500 m/z in high-sensitivity mode with rolling collision energy, were acquired. The 30 most intense precursors were selected for fragmentation per cycle with a dynamic exclusion time of 25 seconds.
The fractions were separated by nano-high performance liquid chromatography (Eksigent Technologies, Dublin, CA, USA) using a secondary RP analytical column (Eksigent, C18, 3 μm, 150 mm × 75 μm). Peptides were subsequently eluted using the following gradient conditions: phase B (98% acetonitrile with 0.1% formic acid) from 5% to 45% B (5–100 minutes). The total flow rate was maintained at 300 nL/minute. An electrospray voltage of 2.3 kV vs the inlet of the mass spectrometer was used. An AB SCIEX TripleTOF™ 4600 mass spectrometer (Foster city, CA, USA) was operated in information-dependent data acquisition mode to automatically switch between MS and MS/MS acquisition. MS spectra were acquired across the mass range of 350–1,250 m/z using an accumulation time of 250 ms per spectrum. MS spectra, scanned from 100 to 1,500 m/z in high-sensitivity mode with rolling collision energy, were acquired. The 30 most intense precursors were selected for fragmentation per cycle with a dynamic exclusion time of 25 seconds.
8
Endogenous Symplekin Purification and Proteomic Analysis
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Endogenous symplekin was purified by IP. Proteins in the gel band were excised and in-gel digested with trypsin. Eluted peptides were subjected to liquid chromatography performed on a nano Acquity UPLC system (Waters Corporation, Milford, MA, USA) connected to a Triple TOF™ 4600 mass spectrometer (AB SCIEX, Framingham, MA, USA). The mass spectra were searched using Mascot Daemon software (Version 2.3.02, Matrix Science, London, UK) based on the UniProtKB/Swiss-Prot database.
9
LC-MS Analysis of Seagrass Extracts
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LC-MS analysis was performed on a Shimadzu HPLC system (controller CBM-20A, two pumps LC-20 AD, a column oven CTO-20 AC and a photo diode array detector SPD-M20A; Shimadzu, Darmstadt, Germany) coupled to a Triple Tof 4600 mass spectrometer (AB Sciex, Canby, USA). The separation of extracted compounds was realised on a Knauer Vertex Plus column (250 × 4 mm, 5 μm particle size, packing material ProntoSIL 120–5 C18-H) with precolumn (Knauer, Berlin, Germany). The column oven temperature was set to 30 °C and 25 μl of undiluted methanolic seagrass extract prepared as described above was injected. The solvent flow rate was 0.8 ml/min. In this time, a gradient was run from 10 to 90% B from minute 0 to 35, 2 min of 90% B, switch to 10% B in 1 min and subsequent equilibration at 10% B for 2 min. Solvent A (water) and B (methanol) were both supplemented with 2 mM ammonium acetate and 0.01% acetic acid. Mass spectra were monitored between 100 and 800 Da in negative ionisation mode. In addition, MS/MS spectra were generated with a collision energy of − 30 eV and measured between 50 and 800 Da. Spectra for the most prominent peaks were compared to database entries in MassBank [35 (link)] and ReSpect [36 (link)] for identification.
10
Quantitative Proteomics with Triple-TOF
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Proteolytic peptides were identified and quantified with a Triple-TOF 4600 mass spectrometer (ABSciex) coupled to the nanoRSLC system (Thermo Fisher Scientific) equipped with a trap column (Acclaim PepMap100C18, 75 μm i.d. × 2 cm, 3 μm) and an analytical column (Acclaim PepMapRSLCC18, 75 μm i.d. × 25 cm, 2 μm, 100 Å).
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