Native MS experiments were carried out on a Synapt G2Si instrument (Waters, Manchester, UK) equipped with a nanoelectrospray ionisation (nESI) source. Mass calibration was performed by a separate infusion of Na iodide cluster ions. Solutions were ionised through a positive potential applied to metal-coated borosilicate capillaries (Thermo Fisher Scientific). Full-length McsB samples (5 μM) were sprayed from 100 mM ammonium acetate, pH 7.85. During the observation of the multimeric state, the following parameters were used: capillary voltage 1.6 kV, sample cone voltage 60 V, extractor source offset 80 V, source temperature 70 °C. Data were processed using MassLynx software version 4.1 (Waters).
Synapt g2si instrument
Manufactured by Waters Corporation
Sourced in United Kingdom, Czechia, United States
The Synapt G2Si is a high-performance mass spectrometer instrument designed for advanced analytical applications. It features a hybrid quadrupole time-of-flight (Q-TOF) architecture, providing accurate mass measurements and high-resolution separation of complex samples. The instrument is capable of performing a variety of mass spectrometry techniques, including tandem MS (MS/MS) and ion mobility separation, enabling comprehensive characterization of analytes.
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Lab products found in correlation
Masslynx software version 4, by Waters Corporation (3 mentions)
Acquity m class nanolc system, by Waters Corporation (2 mentions)
Metal coated borosilicate capillaries, by Thermo Fisher Scientific (1 mentions)
Unifi scientific information system, by Waters Corporation (1 mentions)
Masslynx 4, by Waters Corporation (1 mentions)
Driftscope version 2, by Waters Corporation (1 mentions)
Masslynx v4, by Waters Corporation (1 mentions)
Formic acid, by Merck Group (1 mentions)
Voyager de pro mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Lambda 25 uv vis spectrometer, by PerkinElmer (1 mentions)
8 protocols using synapt g2si instrument
1
Native MS analysis of McsB multimers
2
High-Resolution Mass Spectrometry for Peptide Analysis
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The high-resolution mass spectrometry analysis was performed using a Synapt-G2Si instrument coupled to the Acquity M-class nano-LC system (Waters, Prague, Czech Republic). The HSS T3 column (100 Å, 1.8 µm, 75µm × 250 mm) was used for peptide separation. The column was heated to 40°C. Mobile phase A was composed of 0.1% formic acid in deionized water (v/v), and mobile phase B consisted of acetonitrile with 0.1% formic acid (v/v). The mobile phase gradient program was as follows: 2% B in 0–5 min, 2–40% B in 5–35 min and then 2% B in 35–50 min. The flow rate was 0.3 µL min−1, and the injection volume was 1 µL (final concentration 100 fmol). Data were acquired with MassLynx 4.2 software and analyzed by UNIFI Scientific Information System (Waters, Prague, Czech Republic).
3
N-Glycan Analysis by Ion-Mobility MS
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Prior to ion-mobility electrospray ionisation MS and tandem MS analysis, PNGase F released N-linked glycans were purified on a Nafion® 117 membrane (Sigma-Aldrich) and a trace amount of ammonium phosphate was added to promote phosphate adduct formation. Glycans were analyzed by direct infusion using a Synapt G2Si instrument (Waters) with the following settings: capillary voltage, 0.8–1.0 kV; sample cone, 150 V; extraction cone, 150 V; cone gas, 40 l/h; source temperature, 80 °C; trap collision voltage, 4–160 V; transfer collision voltage, 4 V; trap DC bias, 60 V; IMS wave velocity, 450 m/s; IMS wave height, 40 V; trap gas flow, 2 ml/min; IMS gas flow, 80 ml/min. Data were acquired and processed with MassLynx v4.1 and Driftscope version 2.8 software (Waters).
4
Native Mass Spectrometry of Cascade
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Cascade complex samples were sprayed from in-house prepared gold-coated borosilicate glass capillaries and analyzed on a SYNAPT G2-Si instrument (Waters) as reported before (18 (link)). Briefly, purified Cascade complexes were buffer exchanged to 100 mM ammonium acetate, pH 7 (Sigma) using 3 kDa molecular weight cutoff spin filters (Pall Corp.) and infused to electrospray source at protein concentration of 2–3 μM and the rate around 90 nl/min. To assure best instrument performance in high mass-to-charge range critical parameters were adjusted as follow: source temperature 30°C, capillary voltage 1.7 kV, trap bias voltage 16 V and argon pressure in collision cell (trap) 7 ml/min. Transfer collision energy was kept at constant level of 10 V while trap energy varied between 10–200 V. To obtain accurate mass measurement for individual protein subunits, Cascade samples were dissociated and completely unfolded using a 1% of formic acid (Sigma) in acetonitrile solution mixed with complex in 1:1 ratio. Data analysis was performed in MassLynx software version 4.1 (Waters).
5
Native Mass Spectrometry for NifL Oligomer
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The oligomeric state of NifL was investigated using native mass spectrometry protocol. All experiments were conducted on a SYNAPT G2-Si instrument (Waters) as described previously (70 (link), 71 ). Briefly, the NifL-oxidized samples were buffer exchanged into 250 mM ammonium acetate, pH 8 (Sigma) using 10-kDa molecular weight cutoff spin filters (Pall Corporation) or a 3.5-kDa cutoff slide-a-lyzer minidialysis device (Thermo-Fisher) and infused from in-house prepared gold-coated borosilicate glass capillaries to the electrospray source at a protein concentration of 1 to 5 μM and a flow rate around 90 nL/min. The instrument was tuned to enhance performance in the high mass-to-charge range with the following settings: source temperature 30 °C, capillary voltage 1.2 kV, trap bias voltage 16 V and argon flow in collision cell (trap) 7 mL/min. Transfer collision energy was held at 10 eV while trap energy was varied between 10 and 200 eV. Data analysis was performed in MassLynx software version 4.1 (Waters).
6
High-resolution nanoLC-MS/MS analysis
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The high-resolution mass spectrometry analysis was performed using a Synapt-G2Si instrument coupled to an Acquity M-class nano-LC system and equipped with an ionKey source (Waters Corporation, Milford, MA, USA). A separation device (chip) iKey Peptide BEH (100Å, 18 µm, 150 µm × 100 mm) was used for separation. The iKey device was heated to 40 °C. Mobile phase A was composed of formic acid in MPW (0.1%, v/v), and mobile phase B consisted of acetonitrile with 0.1% formic acid (v/v). A mobile phase gradient program was as follows: 10% B (0–1 min), increasing to 90% B (1–15 min) and then returning to 10% B and re-equilibrating at 17.1 to 20 min. The flow rate was 3 µL·min−1 and the injection volume was 3 µL. In a mass spectrometer, nitrogen was used as a drying gas and high purity nitrogen (N2) was used as a collision gas for collision-induced dissociation (CID). The mass spectrometric parameters were as follows: positive ion mode; desolvation gas flow 800 L·h−1; desolvation temperature 350 °C; capillary voltage 2.8 kV; source temperature 100 °C; mass range, m/z 50–1200 for MS1.
7
Multi-Instrumental Characterization of Samples
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Electrospray ionization mass spectrometric (ESI MS) measurements were carried out using a Waters Synapt G2-Si instrument. ESI MS had a maximum resolution (m/Δm) of 50,000 in the mass range of interest. We used Applied Biosystems Voyager DEPro mass spectrometer for matrix-assisted laser desorption ionization mass spectrometric (MALDI MS) measurements. More details about the measurements are given in Supplementary Notes 2, 3 and 7 . UV/Vis spectra were recorded using a Perkin-Elmer Lambda 25 UV/vis spectrometer. Absorption spectra were typically measured in the range of 200–1100, nm.
8
Lipidomics and Proteomics Analysis
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Lipids were isolated by chloroform/methanol extraction and quantified by ESI-MS/MS [30] [31] [32] .
Sphingolipids were butanol-extracted and analysed by LC-MS/MS [33] [34] [35] . GC-MS Solution Software was used for data acquisition and processing [36] . Urine di-docosahexaenoyl (22:6) bis(monoacylglycerol) phosphate (di-22:6-BMP) content was determined by HPLC-MS [37, 38] .
For LC-MS proteomics, samples were denatured and digested, following by Nanoscale LC separation of tryptic peptides. MS data were acquired in triplicate in ion mobility enabled data independent analysis mode [39] using a Synapt G2-Si instrument (Waters Corporation). ISOQuant was applied for integrated quantitative analysis of data from multiple LC-MS [40, 41] . Reactome, BLAST2GO, GO This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Sphingolipids were butanol-extracted and analysed by LC-MS/MS [33] [34] [35] . GC-MS Solution Software was used for data acquisition and processing [36] . Urine di-docosahexaenoyl (22:6) bis(monoacylglycerol) phosphate (di-22:6-BMP) content was determined by HPLC-MS [37, 38] .
For LC-MS proteomics, samples were denatured and digested, following by Nanoscale LC separation of tryptic peptides. MS data were acquired in triplicate in ion mobility enabled data independent analysis mode [39] using a Synapt G2-Si instrument (Waters Corporation). ISOQuant was applied for integrated quantitative analysis of data from multiple LC-MS [40, 41] . Reactome, BLAST2GO, GO This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
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