For native MS experiments, purified protein constructs were buffer exchanged using Bio-Spin P-6 columns (Bio-Rad) into 0.5 M ammonium acetate. Samples were analysed on the first-generation Synapt mass spectrometer (Waters). Samples were introduced into the mass spectrometer by direct injection method using in house prepared capillaries (borosilicate glass, 1.0 mm × 0.78 mm, Harvard apparatus) created using a needle-puller (P97, Sutter Instruments) and coated with gold using a sputter-coater (SC7620, Emitech) as described previously61 (link). The Synapt instrument was externally calibrated using a 30 mg/mL solution of caesium iodide. Acquisition parameters were as following: capillary voltage 1.2 kV, cone voltage 40 V, extraction cone voltage 1 V, trap/transfer collision energy 6/4 V, bias voltage 4 V and source temperature 40 °C. Mass spectra were analysed using MassLynx software v4.1 (Waters).
Synapt mass spectrometer
Manufactured by Waters Corporation
Sourced in United Kingdom
The Synapt mass spectrometer is a high-resolution, quadrupole time-of-flight (Q-TOF) mass spectrometer designed for a range of analytical applications. It utilizes advanced ion optics and detection systems to provide precise mass measurements and structural information about molecules.
Automatically generated - may contain errors
Lab products found in correlation
Sc7620, by Quorum Technologies (1 mentions)
Bio spin p6 column, by Bio-Rad (1 mentions)
Masslynx software v4, by Waters Corporation (1 mentions)
Ultra performance liquid chromatography (uplc), by Waters Corporation (1 mentions)
Bridged ethyl hybrid c8 column, by Waters Corporation (1 mentions)
Lc qtof ms, by Waters Corporation (1 mentions)
Unify software 1, by Waters Corporation (1 mentions)
Immobilized pepsin column, by Waters Corporation (1 mentions)
Sequencing grade trypsin, by Merck Group (1 mentions)
Nanoacquity uplc, by Waters Corporation (1 mentions)
Glu1 fibrinopeptide b, by Merck Group (1 mentions)
Masslynx v4, by Waters Corporation (1 mentions)
10 protocols using synapt mass spectrometer
1
Native Mass Spectrometry of Purified Proteins
2
Full Protein Mass Determination by ESI-MS
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For full-length mass determination by ESI-MS, the SEC-purified proteins were loaded on a C8 reverse phase column after adding formic acid to 0.1% final concentration and eluted with a gradient of acetonitrile into a Waters Synapt mass spectrometer. For protein identification from gel bands, the SEC-purified gp44 sample was separated by electrophoresis on a 10% Bis–Tris gel. The bands were excised, digested with trypsin, and subjected to LC-MS analysis, using a Thermo Finnigan LTQ XL spectrometer and a Mascot search of the UniRef100 database. Only spectra with a >80% peptide identification probability as determined by Scaffold Viewer 3 were counted for sequence coverage.
3
UPLC-MS/MS Proteomics Workflow
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The sample
was prepared as described
previously.45 (link) LC-MS was performed using
a Waters SYNAPT mass spectrometer outfitted with an ACQUITY UPLC,
an ACQUITY Bridged Ethyl Hybrid C8 column (2.1 × 50 mm2, 1.7 μm particle size, 200 Å; Waters), an ESI ion source,
and a quadruple TOF detector. A gradient of 2–100% aq. MeCN
with 0.1% formic acid (v/v) over 20 min was used.
Fragmentation of the sample was performed using a collision-induced
dissociation (CID) method. A ramping of cone voltage of 15–18
kV during the scan was performed to generate peptide fragments for
MS/MS analysis.
was prepared as described
previously.45 (link) LC-MS was performed using
a Waters SYNAPT mass spectrometer outfitted with an ACQUITY UPLC,
an ACQUITY Bridged Ethyl Hybrid C8 column (2.1 × 50 mm2, 1.7 μm particle size, 200 Å; Waters), an ESI ion source,
and a quadruple TOF detector. A gradient of 2–100% aq. MeCN
with 0.1% formic acid (v/v) over 20 min was used.
Fragmentation of the sample was performed using a collision-induced
dissociation (CID) method. A ramping of cone voltage of 15–18
kV during the scan was performed to generate peptide fragments for
MS/MS analysis.
4
LC-QToF-MS Identification of Phenolic Compounds in Corchorus olitorius Leaf Extract
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Every phenolic compound in the leaf extract of C. olitorius at the optimized microwave-enhanced extraction conditions was identified using LC-QToF-MS (Waters Vion IMS, USA). A C18 column with the dimension of 100 mm × 2.10 mm × 1.80 µm was used. There are two mobile phases used in this analysis; they are water + 0.1% formic acid (A) and 100% acetonitrile (B). The operating conditions were a seal wash period of 5 min, a pre-injector volume of 0 µL, low pressure of 0 psi, high-pressure limit of 18,000 psi, gradient elution was 90% A and 10% B (1.25 min), 45% A and 55% B (4.17 min), 10% A and 90% B (6.25 min), 90% A and 10% B (8.34 min), with the injection volume of 20 µL, the flow rate of 0.50 mL/min, spray voltage of 4 keV, mass range of 100–1000 m/z, sample temperature of 15 °C, column temperature of 40 °C, gas flow of 0.50 mL/min, desolvation gas flow rate of 800 L/h, desolvation temperature of 550 °C, low collision energy of 4 eV, high collision energy ramp between 10 and 45 eV, source temperature of 120 °C. The identifications of phenolic compounds in the leaf extract of C. olitorius were performed by utilising SYNAPT mass spectrometer (Waters) coupled with an electrospray ionization operated in negative ion mode. Waters® UNIFY Software 1.0.0 installed in equipment was used to identify the phenolic compounds.
5
Probing Ubiquitin-Deubiquitinase Complexes
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Complexes between Ub variants and UCH37·RPN13DEUBAD were generated by incubating UCH37·RPN13DEUBAD (60 µM) with the Ub variant (180 µM) for 1 hr on ice in dilution buffer (50 mM HEPES, pH 7.4, 50 mM NaCl, and 1 mM TCEP). Next, 4 µL of protein complex was diluted into either 96 µL of H2O buffer (20 mM sodium phosphate, pH 7.0) for reference mapping or 96 µL of D2O buffer (20 mM sodium phosphate, pD 7.0 in D2O) to initiate deuterium labeling. After 1, 10, 30, 60, and 120 min exposure to the deuterated buffer at 15°C, 50 µL labeling reaction was quenched by mixing with 50 µL quenching buffer (0.8 M Guanidine-HCl, 1% acetic acid, pH 2.2) at 4°C. Quenched samples were immediately injected into the HDX platform. Upon injection, samples were passed through an immobilized pepsin column (2.1 × 30 mm) (Waters Corp.) at 150 µL/min for inline digestion. Following digestion, the resulting peptides were desalted on a 1.8 µm C8 trap column (Waters Corp.) and separated on a 1.0 mm × 5 cm C18 column (Accuity) with linear gradient of 80% B (A is 0.1% formic acid [FA] in H2O, and B is 0.1% FA in acetonitrile) over 12 min, and peptides were identified using ion mobility MS through a Synapt mass spectrometer (Waters Corp.).
6
Mass Spectrometric Analysis of Lipid A
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All the mass spectra of C. sakazakii lipid A samples were obtained from a Waters SYNAPT mass spectrometer, which contains an electrospray ionization (ESI) source. ESI/MS in the negative ion mode was performed to detect lipid A samples, which were dissolved in chloroform (Raetz and Whitfield, 2002 (link)). The instrument was calibrated with sodium formate. ESI/MS was performed at −80 V, and its collisional activation of ions was carried out at −8 V. MassLynx V4.1 software was used to acquire and analysis data.
7
HipA Phosphorylation and Mass Spectrometry
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Freshly purified HipAso and HipAso treated overnight in vitro with 10 mM ATP, 20 mM MgCl2 were diluted 1:50 with 50 mM ammonium bicarbonate buffer. Sequencing-grade trypsin (Sigma) was added in 1:50 ratio to the diluted protein and digested overnight at 37°C. One picomole HipA digestion from those two samples was applied for LCMS analysis on a nanoACQUITY UPLC coupled to a Synapt mass spectrometer used in the Q-TOF mode (Waters). A nanoACQUITY UPLC@2G-V/M Trap column 108 μm * 20 mm 5 μ symmetry C18 (Waters) was used as the trap column at 15 μl/min for 1 min. A nanoACQUITY UPLC 1.8 μm HSS T3 75 μm * 250 mm column (Waters) was used as the separation column at 0.25 μl/min with a gradient as follows: 3% B to 40% B (0–30 min), 40% B to 85% B (30–31 min), 85% B (31–35 min), 85% B to 3% B (35–35.5 min), 3% B until 65 min using 0.1 formic acid as buffer A and 100% acetonitrile as buffer B. All the Nano LCMS data were processed with the PLGS 2.5 software (Waters).
8
High-Resolution ESI-QTOF-MS Analysis of Biomolecules
9
High-Resolution Mass Spectrometry Analysis
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Mass spectrometry was performed using a Waters SYNAPT mass spectrometer equipped with an electrospray ionization (ESI) source. Samples were injected twice: once in positive ESI mode and once in negative ESI mode. The data acquisition range was 100–1500 Da. The lock spray reference scan frequency was 20 s, with a reference cone voltage of 30 V. The MS source temperature was set at 100°C, and the desolvation temperature was 450°C with a gas flow of 900 L/h. The lock mass compound was leucine enkephalin (200 pg/μL), with an m/z of 556.2771 in the positive ion mode and 554.2615 in negative ion mode. The capillary voltages were set to 2.9 kV for ESI+ and 3 kV for ESI−. The cone voltage was 40 kV, and the collision energies were 6 V (trap) and 4 V (transfer), with 2.00 mL/min trap gas flow [16 ].
10
HPLC Analysis and Characterization of 6-Gingerol
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HPLC analysis was performed with empower software version-2, runtime of 20 min by using 0.01M KH acetonitrile as mobile phase A at a flow rate of 1 ml/min, and injection volume of 10.00 µl. Acetonitrile is used to prepare 5 mg/ml of 6-gingerol. Column used in HPLC (Waters -separation module 2695) is made up of intersil ODS-3V with dimensions of 5 mm X 250 mm X 4.6 mm. To evaluate the number and nature of protons present in 6-gingerol (dissolved in DMSO), 1 H NMR (300 MHz) is performed by using bruker avance 300, rheinstetten, Germany. Later on to evaluate the fragmentation pattern and molecular weight of 6-gingerol mass spectroscopy is performed by using synapt mass spectrometer from waters corporation, United Kingdom.
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