Purified recombinant Av-DHDPS and Av-DHDPR were subjected to trypsin digestion and tandem mass spectrometry (MS/MS) sequencing using a Thermo Scientific LTQ Orbitrap Elite ETD Mass Spectrometer as previously reported44 (link)45 (link).
Ltq orbitrap elite etd mass spectrometer
Manufactured by Thermo Fisher Scientific
The LTQ Orbitrap ELITE ETD mass spectrometer is an analytical instrument used for high-resolution, high-accuracy mass spectrometry analysis. It combines the high sensitivity of the LTQ ion trap with the high mass accuracy and resolution of the Orbitrap mass analyzer. The instrument is equipped with Electron Transfer Dissociation (ETD) capability, which enables the analysis of post-translationally modified proteins and peptides.
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5 protocols using ltq orbitrap elite etd mass spectrometer
1
Trypsin Digestion and Mass Spectrometry of Av-DHDPS and Av-DHDPR
2
Trypsin Digestion and Tandem Mass Spectrometry
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The method for trypsin digestion of protein has been described previously (22 (link)). Tandem mass spectrometry analysis was performed using an LTQ Orbitrap ELITE ETD mass spectrometer (Thermo Fisher Scientific). The methods used for liquid chromatography-tandem mass spectrometry (LC-MS/MS) were slightly modified from those described previously (23 (link)). The mass spectrometer was operated in data-dependent acquisition mode in which MS acquisition with a mass range of m/z 400–1000 was automatically switched to MS/MS acquisition under the control of Xcalibur software. The top four precursor ions in the MS scan were selected by Orbitrap, with resolution R = 240 000, and those in the subsequent MS/MS scans, with an ion trap in automated gain control (AGC) mode, where AGC values were 1 × 106 and 1.00 × 104 for full MS and MS/MS, respectively. For fragmentation, electron transfer dissociation was used.
3
Alkylation and Mass Spectrometry Analysis of WT Coa6
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A sample of WTCoa6 (200 μM) was reduced with 4 mM DTT and then alkylated with 50 mM IAA for 30 min before MALDI-TOF. In addition, IAA-labeled and unlabeled samples were subjected to trypsin digestion and tandem MS/MS sequencing using a Thermo Fisher Scientific LTQ Orbitrap Elite ETD Mass Spectrometer as previously reported (56 (link)). Upon digestion, peptides containing the free sulfhydryl groups on the cysteine residues shift by 58 Da because of the alkylation. The predicted mass of alkylated peptides were calculated using PEPTIDEMASS (57 (link)).
4
Trypsin Digestion and Mass Spectrometry
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Proteins were digested with trypsin and tandem mass spectrometry was performed using an LTQ Orbitrap ELITE ETD mass spectrometer (Thermo Fisher Scientific), as described previously (Okuda et al., 2017 (link)).
5
Trypsinization and Tandem Mass Spectrometry
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Trypsinization of proteins was performed as previously described52 (link),53 (link). Tandem MS (MS/MS) analysis was performed using an LTQ Orbitrap ELITE ETD mass spectrometer (Thermo Fisher Scientific). The mass spectrometer was operated in data-dependent acquisition mode, in which MS acquisition with a mass range of 400 to 1000 m/z was automatically switched to MS/MS acquisition under the control of the Xcalibur software (Thermo Fisher Scientific). The top four precursor ions in the MS scan were selected using Orbitrap, with a resolution of R = 240,000, and the ions in subsequent MS/MS scans were analyzed with an ion trap in automated gain control (AGC) mode, in which AGC values were 1 × 106 and 1.00 × 104 for full MS and MS/MS, respectively. Electron transfer dissociation (ETD) was used for fragmentation.
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