LC-(Q) TOF/MS analysis was performed on an UltiMate 3000 system (Dionex, Sunnyvale, CA, USA) comprising a pump, a UV detector (208 nm), and an autosampler cooled to 4 °C with an Inertsil ODS-3v column [5 µm, 4.6 × 250 mm (GL Science Inc., Tokyo, Japan)]. The two samples (each 50 µL) were directly injected into the HPLC column. The separation of tomatines was eluted with 20 mM ammonium acetate/acetonitrile (65:35, v/v) at a flow rate of 700 μL/min at 30 °C, and a MicroQ-TOF III mass spectrometer with an electrospray interface (ESI) source (Bruker Daltonics, Bremen, Germany). The interface voltage and current were 4.50 kV and 1.6 μA for the negative-ion mode. The flow rate of nebulizing gas was 1.5 L/min, and the N2 drying pressure was 0.2 M Pa. The curved desorption line and heat block temperature were both at 200 °C. The detector voltage of the TOF analyzer was 1.68 kV. Ultrahigh-purity argon was used as the collision gas for collision-induced dissociation experiments. The relative energy in collisions was 100%. The sample injection volume was 50 μL. A direct valve was set to transmit and divert the HPLC eluent to waste. Mass spectral data were collected from m/z 160–1100. Data acquisition and processing were carried out with micrOTOFcontrol and dataAnalysis 4.0 (Bruker Daltonics, Bremen, Germany) software.
Microq tof 3 mass spectrometer
Manufactured by Bruker
Sourced in Germany, United States
The MicroQ-TOF III mass spectrometer is a high-performance analytical instrument designed for accurate mass measurements and compound identification. It utilizes quadrupole time-of-flight (Q-TOF) technology to provide precise mass determination and structural elucidation capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Ultimate 3000 system, by Thermo Fisher Scientific (3 mentions)
Microtofcontrol, by Bruker (1 mentions)
Dataanalysis 4, by Bruker (1 mentions)
Tyrosinase t3824, by Merck Group (1 mentions)
L tyrosine t3754, by Merck Group (1 mentions)
Kieselgel 60, by Merck Group (1 mentions)
Rp 18 f254, by Merck Group (1 mentions)
Dithiothreitol (dtt), by GE Healthcare (1 mentions)
Sequencing grade modified porcine trypsin, by Promega (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Zorbax eclipse plus c18, by Agilent Technologies (1 mentions)
H2so4 reagent, by Merck Group (1 mentions)
Kieselgel 60 f254, by Merck Group (1 mentions)
Sephadex lh 20, by GE Healthcare (1 mentions)
Eca 500, by JEOL (1 mentions)
10 protocols using microq tof 3 mass spectrometer
1
LC-QToF/MS Analysis of Tomatines
2
Peptide Sequencing by Nano-ESI MS/MS
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MS/MS of peptides generated by in-gel digestion was performed by nano-ESI on a MicroQ-TOF III mass spectrometer (Bruker Daltonics, Germany) at RT. A potential of 1 kV was applied to the precoated borosilicate nanoelectrospray needles (EconoTipTM, New Objective) in the ion source and combined with a nitrogen back-pressure of 0–5 psi to produce a stable flow rate (10–30 nL/min). The cone voltage was 800 V. The quadrupole analyzer was used to select precursor ions for fragmentation in the hexapole collision cell. Product ions were analyzed using an orthogonal TOF analyzer, fitted with a reflector, a micro-channel plate detector, and a time-to-digital converter. The data were processed using a peptide sequence system.
3
Determining Antioxidant Peptide Characteristics
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Molecular weight (MW) and amino acid sequences of the antioxidant peptide
purified from velvet antler were determined using a MicroQ-TOFIII mass
spectrometer (Bruker Daltonics, Bremen, Germany) coupled with an electrospray
ionization (ESI) source. The purified peptide was dissolved in distilled water
and infused into the ESI source. Its MW was determined by singly charged
(M+H) state analysis in mass spectrum.
purified from velvet antler were determined using a MicroQ-TOFIII mass
spectrometer (Bruker Daltonics, Bremen, Germany) coupled with an electrospray
ionization (ESI) source. The purified peptide was dissolved in distilled water
and infused into the ESI source. Its MW was determined by singly charged
(M+H) state analysis in mass spectrum.
4
Peptide Characterization by Q-TOF MS
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The molecular masses and amino acid sequences of the purified peptides were determined using a quadrupole time-of-flight mass spectrometer (Micro Q-TOF III mass spectrometer, Bruker Daltonics, Bremen, Germany) coupled with an electrospray ionization (ESI) source. The fraction was separately infused into the electrospray source after being dissolved in distilled water containing 0.1% formic acid, and the molecular mass was determined from the doubly charged [M+2H]2 states in the mass spectrum. Following molecular mass determination, peptides were automatically selected for fragmentation, and sequence information was obtained by tandem mass spectrometry (MS) analysis.
5
Chromatographic Separation and Spectroscopic Analysis
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Column chromatography was performed using silica gel (Kieselgel 60, 70–230, and 230–400 mesh, Merck, Darmstadt, Germany) and YMC RP-18 resins (30–50 μm, Fuji Silysia Chemical Ltd., Kasugai, Aichi, Japan). Thin layer chromatography (TLC) was performed using pre-coated silica-gel 60 F254 and RP-18 F254S plates (both 0.25 mm, Merck, Darmstadt, Germany). Compounds were visualized by spraying with 10% aqueous H2SO4 solution and by heating for 2–3 min. NMR spectra were recorded using a JEOL ECA 600 and 400 spectrometeres (Tokyo, Japan), using DMSO-d6 and methanol-d4 as solvents. Mass spectra were measured by Bruker Daltonics MicroQ-TOF III mass spectrometer (Bruker Daltonics, 255748 Germany). Tyrosinase (T3824) and L-tyrosine (T3754) were purchased from Sigma-Aldrich (St. Louis, MO).
6
Acetylation Site Identification of COX2
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To identify the acetylation site of COX2, N-AS (Sigma-Aldrich, 01912)-treated COX2 enzymes (LSBio, LS-G21094) were immediately precipitated with trichroloacetic acid (Merck) and dried. The dried extract was resuspended in 10 μl of 5 M Urea solution and incubated with 1 μg sequencing-grade modified porcine trypsin (Promega) in 0.1 M ammonium bicarbonate buffer at 37 °C for 16 h. The sample was then treated with 1 M DTT (GE Healthcare) at RT for 1 h, followed by alkylation with 1 M iodoacetamide (Sigma-Aldrich) for 1 h. For sequencing, the protein samples were loaded onto a ZORBAX 300SB-C18 column (3.5 μm, 1.0 mm i.d. × 150 mm, Agilent). The column was placed in-line with an UltiMate 3000 system (Dionex, USA) and a splitter system was used to achieve a flow rate of 100 μl min−1. Analytes were eluted with a mobile phase consisting of water: formic acid (100:0.2 v/v) (phase A) and acetonitrile: formic acid (100:0.2 v/v) (phase B) in gradient elution mode for 77 min. Eluted peptides were directly electrosprayed into and MicroQ-TOF III mass spectrometer (Bruker Daltonics, 255748, Germany) by applying 4.5 kV of capillary voltage and a normalized collision energy of 7 eV. The peptides were verified with BioTools 3.2 SR5 (Bruker Daltonics).
7
Peptide Identification in Soybean Genome
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The mobile phase for chromatographic separation consisted of solvent A (0.2% formic acid in water, v/v) and solvent B (0.2% formic acid in acetonitrile, v/v), performed in a gradient manner. Linear gradient elution was performed from 5% B to 95% B in 40 min at a flow rate of 0.2 mL/min. The column compartment was maintained at 30 °C and the injection volume was 5 μL. The analysis was performed using a MicroQ-TOF III mass spectrometer (Bruker Daltonics Inc., Billerica, MA, USA). Additionally, the eluent was monitored by electrospray ion mass spectrometry (ESI-MS) in positive ion mode, and the sample was scanned from m/z 50 to 2000. A homologous peptide search was performed using the Glycine max genome sequence database from a protein BLAST search of the National Center for Biotechnology Information (NCBI) database (www.ncbi.nlm.nih.gov/blast , accessed on 6 April 2020).
8
Antioxidant Peptide Characterization
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Molecular weight and amino acid sequences of antioxidant peptides purified from velvet antler were determined using a MicroQ-TOFIII mass spectrometer (Bruker Daltonics, Hamburg, Germany) coupled with electrospray ionization (ESI) source. The purified peptide was dissolved in distilled water and infused into the ESI source. Its molecular weight was determined by singly charged (M + H) state analysis in mass spectrum.
9
OFBP Characterization by UPLC-Q-TOF MS/MS
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The MS/MS analysis of OFBP was performed through ultra-high performance liquid chromatography-quadrupole time-of-flight (UPLC-Q-TOF) MS/MS at Proteinworks (Daejeon, Republic of Korea) using an Ultimate 3000 system (Dionex; Sunnyvale, CA, USA) and a Micro Q-TOF III mass spectrometer (Bruker Daltonics; Bremen, Germany). The column was Zorbax eclipse plus C18 (3.0 × 100 mm, 1.8 μm, Agilent, Hong Kong, China). The mobile phase consisted of A: H2O/0.1% formic acid and B: acetonitrile/0.1% formic acid with gradient method (flow rate 0.3 mL/min; 0–5 min, 98:2 v/v; 30 min, 80:20 v/v; 31–36 min, 2:98 v/v; 37–45 min, 98:2 v/v).
The presented chromatogram image represents the UV spectrum (red) and the base peak intensity mass chromatogram (blue) area of the mass-produced OFBP. Peptide sequencing was performed on the MS peak corresponding to m/z 543.7632 [M + 2H] at a retention time of 16.4 min, and the results confirmed a 12-amino acid sequence of GASGERGEVGPA (m/z 543.762+; 1085.5086 Da).
The presented chromatogram image represents the UV spectrum (red) and the base peak intensity mass chromatogram (blue) area of the mass-produced OFBP. Peptide sequencing was performed on the MS peak corresponding to m/z 543.7632 [M + 2H] at a retention time of 16.4 min, and the results confirmed a 12-amino acid sequence of GASGERGEVGPA (m/z 543.762+; 1085.5086 Da).
10
Analytical Techniques for Compound Characterization
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High-resolution electrospray ionization mass spectrometry (HR-ESIMS) were recorded on a MicroQ-TOF-III mass spectrometer (Bruker Daltonics, Bremen, Germany). Nuclear magnetic resonance (NMR) experiments were conducted on an ECA500 (JEOL, Tokyo, Japan). Thin layer chromatography analysis was performed on Kieselgel 60 F254 (Merck, Kenilworth, NJ, USA) plates (silica gel, 0.25 mm layer thickness); pure compounds were visualized by dipping plates into 10% (v/v) H2SO4 reagent (Sigma-Aldrich, St. Louis, MO, USA), after which they were heat treated at 300 °C for 30 s. Normal-phase silica gel (Merck 60A, 70–230 or 230–400 mesh ASTM), sephadex LH-20 (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA), and reversed-phase silica gel (YMC Co., Kyoto, Japan, ODS-A 12 nm S-150, S-75 μm) resins were used for open column chromatography. AUDA (10007927), sEH (10011669), and PHOME (10009134) were purchased from Cayman (Cayman, Ann Arbor, MI, USA).
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