Grains of Yukinkomai, Yukinosei and Todorokiwase (100 mg) at 4 DAF were extracted with 8 m urea, 1% (w/v) CHAPS detergent, 10 mm ethylene diamine tetraacetic acid (EDTA) and 5 mm phenylmethylsulfonyl fluoride and centrifuged at 10 000 g for 10 min at 4 °C. The supernatants were precipitated with 10% (w/v) trichloroacetic acid and resolved with 9 m urea, 3% (w/v) IGEPAL detergent, and 2% (v/v) 2‐mercaptoethanol and then used for gel‐based proteomics. The procedures of 2D polyacrylamide gel electrophoresis (2D‐PAGE) and matrix‐assisted laser desorption ionization time‐of‐flight MS (MALDI‐TOF‐MS) were essentially identical to the previous reports (Kaneko et al., 2011 ; Nanjo et al., 2004 ). In 2D‐PAGE, the 1st dimension used isoelectric focusing with ampholine (pH 3.5–10) and the 2nd dimension used sodium dodecyl sulphate (SDS)‐PAGE with 16% separating gel. The 2D gels were stained with Coomassie brilliant blue R‐250 (Nanjo et al., 2004 ). The protein spots excised from the gels were digested by trypsin using standard procedures (Awang et al., 2010 ). MALDI‐TOF‐MS was carried out with a matrix of α‐cyano‐4‐hyrdoxycinnamic acid in an AXIMA‐CFR mass spectrometer (Shimadzu Corp.) and an Autoflex III TOF/TOF mass spectrometer (Bruker BioSpin, Yokohama, Japan; Kaneko et al., 2011 ).
Autoflex 3 tof tof mass spectrometer
Manufactured by Bruker
Sourced in Germany
The Autoflex III TOF/TOF mass spectrometer is a laboratory instrument designed for the analysis of chemical and biological samples. It utilizes time-of-flight mass spectrometry (TOF/TOF) technology to measure the mass-to-charge ratios of ionized molecules. The core function of the Autoflex III is to provide high-resolution and accurate mass analysis of a wide range of analytes, including proteins, peptides, and small molecules.
Automatically generated - may contain errors
Lab products found in correlation
Axima cfr mass spectrometer, by Shimadzu (1 mentions)
Anchorchip, by Bruker (1 mentions)
Flexcontrol 3.0 software package, by Bruker (1 mentions)
Blotglyco, by Sumitomo Bakelite (1 mentions)
Prism 8, by GraphPad (1 mentions)
Mascot software 2, by Matrix Science (1 mentions)
Biotools 3, by Bruker (1 mentions)
Flexanalysis software, by Bruker (1 mentions)
7 protocols using autoflex 3 tof tof mass spectrometer
1
Proteomic Analysis of Rice Grain Developmental Stages
2
Kinetic Analysis of CobB Deacylation Activity
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WT and mutated (Y55F and R58M) CobB were incubated with peptides in the reaction buffer [50 mM tris-HCl, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mM DTT, and 1 mM NAD+ (pH 8.5)] at 37°C for 2 min. The concentrations of the 2-hydroxysiobutyrylated peptide were 2, 5, 8, 10, 12, 16, 32, 40, 60, 142, and 285 μM. One volume of 10% (v/v) trifluoroacetic acid was added to quench the reaction. After cleaning with C18 ZipTips, the resulting peptides were analyzed with an Autoflex III TOF/TOF mass spectrometer (Bruker Daltonics). The measurements were carried out in a reflex positive-ion mode with delayed ion extraction. Before analysis, the instrument was externally calibrated with a mixture of peptide standards. 2,5-dihydroxybenzoic acid (DHB) was used as the matrix for the analysis of labeled/unlabeled peptides. The sample aliquots (1.0 μl) were placed onto a MALDI plate, and then 1.0 μl of the DHB matrix was added and dried at room temperature before MS analysis. An acceleration voltage of 20 kV was used. MS data were analyzed using FlexAnalysis software for spectral processing and peak detection.
3
MALDI-TOF Mass Spectrometry Protocol
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Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis was performed using Autoflex III TOF/TOF mass spectrometer (Bruker Daltonics, Leipzig, Germany) (mass tolerance: ≤4 ppm). The measurements were conducted in reflex positive-ion mode with delayed ion extraction. Prior to analysis, the instrument was externally calibrated with a mixture of peptide standards. 2,5-Dihydroxybenzoic acid (DHB) was used as the matrix for the analysis of peptides. Sample aliquots of 1.0 µl were placed onto the MALDI plate. Then, 1.0 µl of the DHB matrix was added and dried at room temperature. MS data were analyzed using Flexanalysis software (3.3.65.0) for spectral processing and peak detection.
4
Molecular Mass Determination of Enterocin LNS18
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The molecular mass of enterocin LNS18 was confirmed by mass assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). The sample was prepared at a 1:1 dilution, with a matrix composed of (3 mg/mL α-cyano-4-hydroxycinnamic acid; CHCA) in 50% (v/v) acetonitrile, and 1% (v/v) formic acid before spotting on an Anchor Chip (Bruker- Daltonics, Bremen, Germany) and dried at RT then analyzed with an Autoflex III TOF/TOF mass spectrometer (Bruker-Daltonics).
5
MALDI-TOF-MS Analysis of IgG Glycosylation
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Samples for MALDI-TOF-MS were prepared from 10 μg of purified IgGs from CIA-induced ST6Gal1f/f AID-Cre, ST6Gal1f/f, AID-Cre and C57BL/6j mice and total serum IgG from ST6Gal1 KO mice by BlotGlyco (Sumitomo Bakelite Co., Tokyo, Japan) according to the manufacturer's protocol55 (link). Samples were analysed by MALDI-TOF system by using an Autoflex III TOF/TOF mass spectrometer equipped with a reflector and controlled by the FlexControl 3.0 software package (Bruker Daltonics GmbH, Bremen, Germany). Details of the method used for MALDI-TOF-MS analysis are described in Supplementary Methods .
6
Enzymatic Peptide Modification Kinetics
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This method was described previously8 (link). CobB was incubated with modified peptides (AETAEK(la)YGDEQVK or AETAEK(ac)YGDEQVK, from GpmA) in the CobB reaction buffer at 37 °C for 2 min. Meanwhile, YiaC was incubated with unmodified peptide (TICHDKAFVK, from PncB) in the YiaC reaction buffer. The concentrations of the peptides were 5, 10, 16, 32, 60, and 285 uM, and reaction time were 1, 5, 15, and 30 min. After quenching the reaction by adding equal volume of 10% (v/v) trifluoroacetic acid and cleaning with C18 ZipTips, the products were analyzed by Autoflex III TOF/TOF mass spectrometer (Bruker Daltonics) with a reflex positive-ion mode equipping delayed ion extraction. 0.1 μl samples mixed with 2,5-dihydroxybenzoic acid (DHB) were analyzed by MS. An acceleration voltage of 20 kV was used. MS data were analyzed using FlexAnalysis software for spectral processing and peak detection. The enzyme kinetics were analyzed by Prism GraphPad 8.0 software.
7
MALDI-TOF Mass Spectrometry Protein Identification
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Samples were analysed with an Autoflex III TOF/TOF mass spectrometer (Bruker-Daltonics, USA). Typically, 1000 scans for peptide mass fingerprinting (PMF) and 2000 scans for MS/MS were collected. Automated analysis of mass data was performed using FlexAnalysis software (Bruker-Daltonics, USA). Internal calibration of MALDI-TOF mass spectra was performed using two trypsin autolysis ions with m/z 842.510 and m/z 2211.105; as for MALDI-MS/MS, calibrations were performed with a fragment ion spectrum obtained from the proton adducts of a peptide mixture covering the m/z 700-4000 region. The typical error observed in mass accuracy for calibration was usually below 50 ppm. MALDI-MS and MS/MS data were combined through the BioTools 3.0 program (Bruker-Daltonics, USA) to interrogate the NCBI non-redundant protein database SwissProt 2014_03 (542782 sequences; 193019802 residues) using MASCOT software 2.3 (Matrix Science, UK). Relevant search parameters were set as follows: enzyme, trypsin; fixed modifications, carbamidomethyl (C); oxidation (M); 1 missed cleavage allowed; peptide tolerance, 50 ppm; MS/MS tolerance, 0.5 Da.
Peptide mass fingerprinting and fragmentation by MS-MALDI-TOF was carried out in the Proteomics and Genomics Facility (CIB-CSIC), a member of ProteoRed-ISCIII network.
Peptide mass fingerprinting and fragmentation by MS-MALDI-TOF was carried out in the Proteomics and Genomics Facility (CIB-CSIC), a member of ProteoRed-ISCIII network.
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