GTEs were subjected to LC–MS analysis with an IT-TOF instrument (Shimadzu, Kyoto, Japan) as previously described42 (link). Brifely, the equipment was fitted with a Luna C18(2) column (250 mm × 1.0 mm, 5 μm particle size, Phenomenex, Torrance, CA) maintained at 40°C. The mobile phase solvents were 0.05% aqueous formic acid (solvent A) and 0.05% formic acid in methanol (solvent B). Solvent B was increased from 5% to 60% over 7.5 min. Solvent B was then increased from 60% to 100% at 10.1 min. Flow rate was kept constant (0.1 mL/min).
It tof instrument
Manufactured by Shimadzu
Sourced in Japan
The IT-TOF instrument is a high-performance mass spectrometer that uses ion trap and time-of-flight (TOF) technologies to analyze a wide range of samples. It provides high-resolution, accurate-mass measurement of ions, enabling precise identification and characterization of chemical compounds.
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Lab products found in correlation
4 protocols using it tof instrument
1
Quantifying metabolites using LC-MS
2
Non-targeted LC-MS Analysis of Green Tea
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Green tea extracts were diluted 1:10 with distilled water and subjected to non-targeted LC–MS analysis with an IT-TOF instrument (Shimadzu, Kyoto, Japan). The equipment was fitted with a L-column 2 ODS (2.1 mm I.D. × 150 mm, 3 μm; CERI, Saitama, Japan) maintained at 40 °C. The mobile phase solvents were: solvent A, H2O (0.05% formic acid) and solvent B, acetonitrile (0.05% formic acid). The gradient program was as follows: 0–2 min (A:B 95:5, v/v), 2–3.5 min (80:20, v/v), 3.5–10 min (60:40, v/v), 10–13 min (0:100, v/v), 13–15.5 min (0:100 v/v), 15.5–16 min (95:5 v/v), 16–20 min (95:5 v/v). The flow rate was 0.15 mL/min and the injection volume was 5 μL. The MS parameters were: electrospray ionization source in positive and negative modes with survey scans at m/z 100–700, nebulizing gas flow rate of 1.5 L/min, drying gas pressure of 0.2 MPa, capillary voltage of + 4.5/–3.5 kV, CDL temperature of 200 °C and heat block temperature of 200 °C.
3
Peptide Purification and Characterization
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After release from the resin, the crude peptide products were analyzed using a Thermo Separation HPLC system with UV detection (210 nm) on a Vydac Protein RP C18 column (4.6 × 250 mm, 5 μm), with a gradient elution of 0%–80% S2 in S1 (S1 = 0.1% aqueous TFA; S2 = 80% acetonitrile + 0.1%) for 40 min (flow rate 1 mL/min). TEE was analyzed on an Aeris Peptide XB-C18 column (50 mm × 2.1 mm; 3.6 μm bead diameter) using a Shimadzu IT-TOF instrument equipped with a PDA detector. The separation conditions were similar to those described in section 2.3. The main carbonylated product was purified using a preparative reversed-phase HPLC on a Vydac C18 column (22 mm x 9 250 mm), using solvent systems: S1 0.1% aqueous TFA, S2 80% acetonitrile + 0.1% TFA, linear gradient from 50 to 100% of S2 for 55 min, flow rate 7.0 ml/min, UV detection at 210 and 280 nm. The resulting fractions were collected and subjected to lyophilisation. The identities of the products were confirmed by MS analysis, using an Apex Ultra FT-ICR (Bruker, Germany) mass spectrometer equipped with an electrospray (ESI) ionization source.
4
LC-MS/MS Protein Identification Protocol
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LC-MS/MS analyses of the samples were performed on a SHIMADZU IT-TOF instrument using automatic fragmentation mode. The spectra were analysed by a bioinformatic approach using the LabSolution software to convert LC data file to mzXML format. Data were analysed using PEAKS search engine. For this purpose, we downloaded a FASTA file from the UniProt database. The following search parameters were set: precursor mass error tolerance ±0.1 Da; fragment ion tolerance ±0.2 Da; CID fragmentation, semi-specific digestion mode by trypsin, assuming up to three missed cleavages per peptide. Moreover, carbamidomethylation, as a fixed post-translational modification (PTM), was chosen. Deamidation, dehydration, oxidation of methionine, and hexose modifications were set as variable PTMs and three maximum variable PTMs per peptide were allowed. A false discovery rate (FDR) < 2% at the spectra level was used to filter search results. Additionally, the presence of at least two unique peptides was set for the identification of the corresponding protein. The obtained results, especially the statistical data, were reported into the Supporting Information .
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