Sacculi samples obtained by boiling bacterial biomass in 5% SDS were used for peptidoglycan isolation106 (link),107 (link). Two treatments were tested before muramidase digestion: (i) DNase, RNase, α-amylase, and α-chymotrypsin alone or (ii) with addition of LiCl, EDTA, and HF treatments after protease inactivation. The soluble fraction obtained from muramidase digestion was reduced using 0.5 M sodium borate (pH 9.5) and sodium borohydride (10 mg/ml) and the pH was adjusted to 3.5 with phosphoric acid for liquid chromatography (LC). Solubilized muropeptides were detected and analysed by LC-MS using UPLC coupled with a Xevo G2/XS Q-TOF mass spectrometer (Waters Corp.) operated in positive ionization mode108 (link). Data were acquired and processed using the UNIFI software package (Waters Corp.). The molecular structure of muropeptides included in the UNIFI compound library was obtained using ChemSketch (www.adclabs.com ).
Unifi software package
Manufactured by Waters Corporation
Sourced in United Kingdom
UNIFI software package is a comprehensive data management and analysis solution developed by Waters Corporation. It provides a centralized platform for acquiring, processing, and managing data generated by various analytical instruments. The core function of UNIFI is to enable efficient data management and streamline the data analysis workflow for researchers and scientists working in various industries, including pharmaceuticals, biotechnology, and academia.
Automatically generated - may contain errors
3 protocols using unifi software package
1
Isolation and Analysis of Bacterial Peptidoglycan
2
LC-HRMS Analysis of Mouse Urine Metabolites
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LC-HRMS analysis was
performed on a Waters Synapt XS HDMS coupled
with an ACQUITY UPLC I-Class system. Separation was carried out on
the Acquity Premier CSH C18 column (150 mm × 2.1 mm, 1.7 μm).
Mouse urine (25 μL) was diluted (10×) with solvent A. The
separation was performed using a binary gradient with 0.1% formic
acid in UHPLC-grade water (Honeywell) as solvent A and 0.1% formic
acid in acetonitrile as solvent B (UHPLC–MS, Thermo Scientific).
Gradient conditions: 0.0–11.0 min, 100–77% A; 11.0–14.6
min, 77–5% A; 14.6–17.0 min, 5% A; and 17.05–20.0
min, 100% A. The following settings were used: flow rate, 0.5 mL/min;
sample injection volume, 1 μL; column temperature, 50 °C;
sample temperature, 5 °C. Synapt XS HDMS data were acquired in
the negative ion MSe mode. Authentic standards of 3HPMA and 23HPMA
in water (100 ng/mL) were also prepared and analyzed using identical
conditions to confirm and validate the assignments in urine samples
(retention time, MS/MS, and external database match). The Waters UNIFI
software package was used for data analysis and metabolite identification.
The El-Maven and PollyPhi packages (Elucidata, MA) were used to assign 13C-labeled isotopologues, as well as to correct for the natural
abundance of 13C, as described previously.37 (link)
performed on a Waters Synapt XS HDMS coupled
with an ACQUITY UPLC I-Class system. Separation was carried out on
the Acquity Premier CSH C18 column (150 mm × 2.1 mm, 1.7 μm).
Mouse urine (25 μL) was diluted (10×) with solvent A. The
separation was performed using a binary gradient with 0.1% formic
acid in UHPLC-grade water (Honeywell) as solvent A and 0.1% formic
acid in acetonitrile as solvent B (UHPLC–MS, Thermo Scientific).
Gradient conditions: 0.0–11.0 min, 100–77% A; 11.0–14.6
min, 77–5% A; 14.6–17.0 min, 5% A; and 17.05–20.0
min, 100% A. The following settings were used: flow rate, 0.5 mL/min;
sample injection volume, 1 μL; column temperature, 50 °C;
sample temperature, 5 °C. Synapt XS HDMS data were acquired in
the negative ion MSe mode. Authentic standards of 3HPMA and 23HPMA
in water (100 ng/mL) were also prepared and analyzed using identical
conditions to confirm and validate the assignments in urine samples
(retention time, MS/MS, and external database match). The Waters UNIFI
software package was used for data analysis and metabolite identification.
The El-Maven and PollyPhi packages (Elucidata, MA) were used to assign 13C-labeled isotopologues, as well as to correct for the natural
abundance of 13C, as described previously.37 (link)
3
Nanoflow LC-IMS-MS Protocol for Vesicle Analysis
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Purified vesicles were analysed by ultra-performance nanoLC (ACQUITY M Class, Waters) coupled to an IMS mass spectrometer (SYNAPT G2-Si, Waters) fitted with a NanoLockSpray source (Waters). Samples were loaded via a Symmetry C18 5 µm, 180 µm × 20 mm trap column (Waters) and separated through a HSS T3 C18 1.8 µm, 75 µm × 150 mm analytical column (Waters). Samples were separated using a 5 to 95% acetonitrile 0.1% formic acid gradient over 35 min at a flow rate of 300 nL/min. The mass spectrometer was operated in positive ion mode with a capillary voltage of 3.0 kV, cone voltage of 40 V and a source offset of 80 V. Before analysis the instrument was calibrated with NaI and during analysis a LockMass reference, Glu-1-fibrinopeptide B (Waters), was delivered to the NanoLockSpray source. Mass spectra were collected, over 50–2000 m/z, alternating between low (4 eV) and elevated (15–45 eV) collision energies at a scan speed of 1.0 s. Data were processed using the UNIFI software package from Waters (Wilmslow, U.K.).
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