Samples containing 2.5 nmol protein were first alkylated and digested with LysC and trypsin for 4 hours, then they were analyzed using a Dionex UltiMate 3000 RSLCnano system (Sunnyvale, CA, USA) coupled with a Maxis II QTOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany). Protein identification, quantitation was performed using the Byonic v3.8.18 (https://proteinmetrics.org ) and MaxQuant v1.6.17 (https://maxquant.org ) software respectively. Detailed methodology – including missing data imputation and differential expression statistics – is described in the methodological supplementary.
Maxis 2 q tof mass spectrometer
Manufactured by Bruker
Sourced in United States, Germany
The MaXis II Q-TOF mass spectrometer is a high-performance, quadrupole-time-of-flight mass analyzer developed by Bruker. It is designed to provide accurate mass measurements and high-resolution analysis of a wide range of molecular compounds.
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5 protocols using maxis 2 q tof mass spectrometer
1
Shotgun Proteomics Workflow Analysis
2
LC-MS Analysis of Crosslinking Mixtures
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LC/MS analyses were performed using
an Agilent 1100 series LC coupled to a MicrOTOF-Q (Bruker Daltonics,
Bremen, Germany) or to a maXis II Q-TOF mass spectrometer (Bruker).
The mass spectrometer was operated in positive mode with a capillary
voltage of 4500 V. Acquisitions were performed on the mass range of
200–1850 m/z. Calibration
was performed using the singly charged ions produced by a solution
of Tune mix (G1969–85000, Agilent, U.S.A.). Data analysis was
performed by using Compass DataAnalysis 4.3 (Bruker Daltonics). A
cross-linking reaction mixture containing GSH andPDO 2 (or probe 9 ) was directly analyzed onto a HPLC connected
to MicrOTOF-Q. Compounds were separated on a XBridge Peptide BEH C18
column (300 Å, 3.5 μm, 2.1 mm × 250 mm) column. The
gradient was generated at a flow rate of 250 μL/min using 0.1%
trifluoroacetic acid (TFA) in water for mobile phase A and ACN containing
0.08% TFA for mobile phase B at 60 °C. Phase B was increased
from 5 to 85% in 45 min.
an Agilent 1100 series LC coupled to a MicrOTOF-Q (Bruker Daltonics,
Bremen, Germany) or to a maXis II Q-TOF mass spectrometer (Bruker).
The mass spectrometer was operated in positive mode with a capillary
voltage of 4500 V. Acquisitions were performed on the mass range of
200–1850 m/z. Calibration
was performed using the singly charged ions produced by a solution
of Tune mix (G1969–85000, Agilent, U.S.A.). Data analysis was
performed by using Compass DataAnalysis 4.3 (Bruker Daltonics). A
cross-linking reaction mixture containing GSH and
to MicrOTOF-Q. Compounds were separated on a XBridge Peptide BEH C18
column (300 Å, 3.5 μm, 2.1 mm × 250 mm) column. The
gradient was generated at a flow rate of 250 μL/min using 0.1%
trifluoroacetic acid (TFA) in water for mobile phase A and ACN containing
0.08% TFA for mobile phase B at 60 °C. Phase B was increased
from 5 to 85% in 45 min.
3
Diglycine Modification Detection via ETD
4
Molecular Formula Identification via UHPLC-QTOF-MS
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To acquire molecular formulae information, samples were analysed using an Ultra‐high‐resolution MaXis II Q‐TOF mass spectrometer equipped with electrospray source coupled with Dionex 3000RS UHPLC was employed (Bruker). A reverse phase C18 column (Agilent Zorbax, 100 x 2.1 mm, 1.8 μm) and a guard column (Agilent C18, 10 x 2.1 mm, 1.8 μm) were used for separation applying a linear gradient of 95:5 A/B to 0:100 A/B over 20 min (Mobile phase A: water with 0.1% formic acid, B: acetonitrile with 0.1% formic acid). The injected volume was 2 μl, and the flow rate was 0.2 ml min‐1. At the beginning of each run, 7.5 μl of 10 mM of sodium formate solution was injected for internal calibration. The mass spectrometer was operated in positive ion mode with a 50–2500 m/z scan range. MS/MS data were acquired for the three most intense peaks in each scan.
5
Characterizing SST Strains via HR-MS/MS
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High resolution tandem mass spectrometry (HR-MS/MS) data for the four SST strains was generated using Agilent 1290 Infinity UHPLC coupled to a Bruker MAXIS II Q-ToF mass spectrometer. LC separations utilized a Phenomenex Kinetex XB-C18 (2.6 µM, 100 × 2.1 mm) column. MS and MS2
experiments were carried out in positive ionization modes using a MS and MS2 range of m/z 100-2000.
HR-MS/MS raw data files were converted from .baf to .mzXML using MSConvert and a molecular network was created using Global Natural Products Social (GNPS) Molecular Networking (Wang et al., 2016) . GNPS parameters for creating the molecular network were not changed from the standard parameters GNPS recommends except for the minimum peak intensity, which was set to 5% in order to remove any potential artifacts from interfering in the analysis.
experiments were carried out in positive ionization modes using a MS and MS2 range of m/z 100-2000.
HR-MS/MS raw data files were converted from .baf to .mzXML using MSConvert and a molecular network was created using Global Natural Products Social (GNPS) Molecular Networking (Wang et al., 2016) . GNPS parameters for creating the molecular network were not changed from the standard parameters GNPS recommends except for the minimum peak intensity, which was set to 5% in order to remove any potential artifacts from interfering in the analysis.
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