All data were acquired using a Waters Xevo QTOF mass spectrometer (Milford, MA, usa). A μ-PrepCell thin-layer electrochemical flow cell (Antec BV, Leyden, The Netherlands) was coupled with the mass spectrometer by liquid sample DESI (Scheme S-1, Supporting Information ), which was previously described in details [27 (link)]. The electrochemical flow cell was equipped with a magic diamond electrode (12 mm × 30 mm, Antec BV) as the working electrode (WE), and a Roxy potentiostat (Antec BV) was employed to apply a reduction potential to the WE. A sample syringe was connected with the cell by a piece of PEEK capillary tube. The sample solution was injected at a flow rate of 5 μL/min and flowed through the cell for electroreduction. The reduced species flowed out of the cell via a piece of fused silica capillary and underwent ionization by DESI via the interaction with charged microdroplets from DESI spray. H2O:MeOH (v/v 50:50) containing 1% FA was used as the DESI spray solvent and injected at a flow rate of 5 μL/min. A high voltage of +5 kV was applied to the DESI spray probe with nebulization gas of N2 (pressure 170 psi). Collision induced dissociation (CID) was carried out for ion structural analysis, in which a wide mass selection window was used for simultaneous selection of both DSP-d0 and DSP-d8 labeled cross-link ions or a pair of the reduced cross-link ions.
Xevo qtof mass spectrometer
Manufactured by Waters Corporation
Sourced in United States
The Xevo QTOF mass spectrometer is a high-resolution, accurate-mass instrument designed for a wide range of analytical applications. It utilizes quadrupole time-of-flight (QTOF) technology to provide precise mass measurements and structural elucidation capabilities.
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Lab products found in correlation
Acquity uplc beh c18 column, by Waters Corporation (2 mentions)
Acquity uplc system, by Waters Corporation (2 mentions)
Beh c18 column, by Waters Corporation (1 mentions)
Nanoacquity liquid chromatography system, by Waters Corporation (1 mentions)
Diclofenac sodium, by Merck Group (1 mentions)
Sulfanilamide, by Merck Group (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharide lps salmonella serotype, by Merck Group (1 mentions)
N 1 1 napthyl ethylenediamine dihydrochloride, by Merck Group (1 mentions)
Ascend 400 mhz spectrometer, by Bruker (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ibuprofen, by Merck Group (1 mentions)
Uplc system, by Waters Corporation (1 mentions)
Hitrap desalting, by GE Healthcare (1 mentions)
Acquity ultra performance lc system, by Waters Corporation (1 mentions)
Uplc beh c18 1.7 μm column, by Waters Corporation (1 mentions)
Acquity uplc, by Waters Corporation (1 mentions)
Uv 2550, by Shimadzu (1 mentions)
Avance 600 mhz spectrometer, by Bruker (1 mentions)
19 protocols using xevo qtof mass spectrometer
1
Electrochemical DESI-MS Analysis of Cross-links
2
Heparin Digests Analysis by LC-MS
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The liquid chromatography/mass spectrometry (LC/MS) method used to analyze heparin digests was as described previously (8 (link)), and used an Acquity UPLC BEH C18 column, 150 × 2.1 mm, 1.7 μm (Waters SAS, En Yvelines Cedex, France). Mobile phase A was water, and mobile phase B water:acetonitrile (30:70). The ion pairing reagent, heptyl amine (HPTA; 7.5 mM), and a buffering agent, hexafluroisopropanol (50 mM), were added to both A and B. A linear gradient (t0min B% 1; t70min B% 70) was applied for elution at a flow rate of 0.22 mL/min. Column temperature was set at 30°C and diode array detection used. Double UV detection was performed at 265 nm and 232 nm. Electrospray ionization mass spectra were obtained using a Waters Xevo Q-Tof mass spectrometer. The electrospray interface was set in negative ion mode with a capillary potential of 2000 V and a sampling cone potential of 20 V. Source and desolvation temperatures were 120 and 300°C, respectively. Nitrogen was used as the desolvation (750 L/min) and cone (25 L/min) gas. The mass range was 50–2500 Da (scan rate = 0.8 s). Acquisition was performed in MSE mode with low energy at 7 V and a high energy ramp from 30 to 50 V.
3
Profiling Metabolites by UPLC-QTOF-MS
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Samples were analyzed on a Waters Xevo Q-TOF Mass Spectrometer coupled to a Waters Acquity UPLC system using a Waters BEH C18 column 50x2.1mm 1.6 μm. Analysis was done in either acidic (A: H2O + 0.1% formic acid, B: ACN + 0.1% formic acid) or basic eluents (A: H2O + 0.1% NH3 solution, B: ACN + 0.1% NH3 solution). The same gradient was used with both types of eluent: 98% A for 0.5 min then linear gradient to 65% A (3.5 min) then to 5% A in 1 min, gradient held for 1 min before a step change back to the to the starting conditions and an equilibration time of 1 min (total run time 7 min).
The mass spectrometer was operated in both positive (Capillary Voltage 2.3 kV) and negative mode (Capillary Voltage 1.5 kV). Source temperature and desolvation gas temperatures were constant at 120°C and 500°C respectively. In MS operation, spectra were acquired every 0.2 s over a 50–1000 amu range.
The mass spectrometer was operated in both positive (Capillary Voltage 2.3 kV) and negative mode (Capillary Voltage 1.5 kV). Source temperature and desolvation gas temperatures were constant at 120°C and 500°C respectively. In MS operation, spectra were acquired every 0.2 s over a 50–1000 amu range.
4
Heparin Structural Characterization by Ion-Pair UPLC-MS
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Heparin digests were injected on ion-pair LC/MS chromatography using Acquity UPLC BEH C18 column, 2.1 × 150 mm, 1.7 μm (Waters). Mobile phase A was water, and mobile phase B water/acetonitrile (30:70). The ion pairing reagent, HPTA (7.5 mM) and a buffering agent, HFIP (50 mM) were added to both A and B. A linear gradient (t0 min B% 1; t 60 min B% 60) was applied for elution at a flow rate of 0.22 mL/min. Column temperature was set at 30 °C and diode array detection used. Double UV detection was performed at 265 nm and 232 nm. An NRE building block-specific signal was obtained by the reconstruction of 265 nm − 2.6 × 232 nm.
Electrospray ionization (ESI) mass spectra were obtained using a Waters Xevo Q-Tof mass spectrometer. The electrospray interface was set in negative ion mode with a capillary voltage of 2000 V and a sampling cone voltage of 20 V. The source and the desolvation temperatures were 120 °C and 300 °C, respectively. Nitrogen was used as desolvation (750 L/min) and cone gas (25 L/min). The mass range was 50–2500 Da (scan rate = 0.8 s). Acquisition was performed in MSE mode [29 ] with low energy at 7 V and a high energy ramp from 30 V to 50 V.
Electrospray ionization (ESI) mass spectra were obtained using a Waters Xevo Q-Tof mass spectrometer. The electrospray interface was set in negative ion mode with a capillary voltage of 2000 V and a sampling cone voltage of 20 V. The source and the desolvation temperatures were 120 °C and 300 °C, respectively. Nitrogen was used as desolvation (750 L/min) and cone gas (25 L/min). The mass range was 50–2500 Da (scan rate = 0.8 s). Acquisition was performed in MSE mode [29 ] with low energy at 7 V and a high energy ramp from 30 V to 50 V.
5
Nanoscale Proteomics Analysis Protocol
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The trypsin digested samples were analyzed using a Waters Xevo QTOF mass spectrometer equipped with a nanospray source, coupled to a Waters NanoAcquity liquid chromatography system. The samples were injected onto an Acquity UPLC Symmetry C18 10 K 2 g V/M trapping column, and eluted onto an Acquity UPLC peptide BEH C18 column 130 A, 1.7, 75 µm × 250 mm. In-line fractionation was done using a two-phase gradient system. Solvent A was 2% acetonitrile (ACN) in 0.1% formic acid and (solvent B)100% ACN was 0.1% formic acid. The mass spectrum acquisition was done in positive-ion survey mode using a data-dependent method. The mass acquisition window was set for 100 −1990m/z in full MS mode. The detector was set to positive ions in continuum mode. An intensity threshold was set for 50 counts /sec with a scan time of 0.9 sec. MS/MS mode was set with a mass window of 50–1990 m/z. Three MSMS scans were collected for each MS peak with charge state peak selection from +1 to +4. The collision energy was ramped according to charge.
6
Detailed Analytical Protocols for Cell-Based Assays
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Bovine serum albumin, lipopolysaccharide (LPS) (Salmonella serotype), N-(1-1-napthyl) ethylenediamine dihydrochloride, resazurin sodium 10%, streptomycin, sulfanilamide, tetra methyl benzidine (TMB), ibuprofen, diclofenac sodium and Trypan blue were purchased from Sigma-Aldrich (Castle Hill, Australia). Dulbecco’s modified Eagle’s medium (DMEM), Phosphate buffer saline (PBS) foetal bovine serum (FBS) and glutamine were GIBCO brands purchased from Life Technologies (Mulgrave, Australia). Recombinant IFN-γ and TNF-α ELISA kits were purchased from PeproTech Asia (Rehovot, Israel). Ninety-six-well cell culture plates were initially from Greiner Bio-One, but were later replaced by Eppendorff plates as they show improved evaporation characteristics (no edge effect).
NMR spectra were recorded on a Bruker Ascend 400 MHz spectrometer (Bruker Biospin GmbH, Bremen, Germany), in the solvents indicated and referenced to residual 1H signals in deuterated solvents. HRMS was carried out using a Waters Xevo Q-TOF mass spectrometer operating in the positive ESI mode.
NMR spectra were recorded on a Bruker Ascend 400 MHz spectrometer (Bruker Biospin GmbH, Bremen, Germany), in the solvents indicated and referenced to residual 1H signals in deuterated solvents. HRMS was carried out using a Waters Xevo Q-TOF mass spectrometer operating in the positive ESI mode.
7
High-Resolution LC-MS Metabolomics Analysis
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LC-MS was performed using a Waters Xevo QTOF mass spectrometer equipped with an ACQUITY UPLC system. The heated capillary and spray voltage were maintained at 300 °C and 3.0 kV, respectively. Nitrogen was used at 80 psi for the sheath gas and 20 psi for the auxiliary gas. Full scan mass spectra in the range m/z 100–5000 were acquired in positive ion mode with a scan speed of one scan per second. The tandem mass spectrometry collision gas was helium with a collision energy of 30% of the 5 V end-cap maximum ticking voltage.
8
Phosphorylation of Alpha-synuclein by PLK2
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Alpha-synuclein (Abcam #ab51189) was phosphorylated using PLK 2 (Invitrogen Cat# PV4204) at a concentration of 1.44 mg/ml (100 μM). The phosphorylation reactions were carried out in the presence of 1.09 mM ATP, 1× reaction solution (2 mM HEPES, 10 mM MgCl2, 2 mM DDT, pH 7.4) and 1 μg of PLK/144 μg/ml of α-syn at 30 °C for 24 h. The reaction was quenched with 25 mM EDTA. After quenching the sample was desalted on a G25 column (HiTrap Desalting, GE healthcare #G-25 17–1408-01) into DPBS (Invitrogen #14190–094).
The sample was analyzed by LC-MS. Briefly; the sample was separated on a C4 2.1 × 50 mm BEH300 column run in FA/ACN and introduced to a XEVO QTOF Mass spectrometer (Waters). The multicharged signal obtained from the ion trace was deconvoluted and the mass identified to be a mixture of a non-phosphorylated 14,459 Da α-synuclein (minor component) and the major 14,539 Da phosphorylated species corresponding to a + 80 Da change caused by phosphorylation on serine 129.
The sample was analyzed by LC-MS. Briefly; the sample was separated on a C4 2.1 × 50 mm BEH300 column run in FA/ACN and introduced to a XEVO QTOF Mass spectrometer (Waters). The multicharged signal obtained from the ion trace was deconvoluted and the mass identified to be a mixture of a non-phosphorylated 14,459 Da α-synuclein (minor component) and the major 14,539 Da phosphorylated species corresponding to a + 80 Da change caused by phosphorylation on serine 129.
9
LC-MS/MS Analysis of Methanol Samples
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Samples were prepared in methanol at a concentration of 2 mg/mL and 5 µl were injected for each LC-MS analysis. LC-MS/MS analyses were carried out on a Waters Acquity UPLC system equipped with a Waters Xevo™ QToF mass spectrometer and an electrospray source. Mass spectrometry (MS) data were acquired simultaneously in the positive mode at a mass range of m/z 1000–1000 Da. Samples were dissolved in methanol at the concentration of 2 mg/mL and injected into a 5 μm SunFire™ C-18 column (250 × 4.6 mm). Two solvents, water + 0.1 % acetic acid (solvent A) and methanol + 0.1 % acetic acid (solvent B) were used as the mobile phase at a flow rate of 0.4 mL/min. The ESI conditions and mass spectrometry acquisition parameters were set as follows: capillary voltage, detector voltage, sampling cone, and extraction cone voltages: 3.0 kV, 2.2 kV, 25 V, and 4.0 V respectively; source and desolvation temperatures: 150 and 250 oC, respectively and cone and desolvation flow: 50 and 600 L/h, respectively. Eight most intense ions with a threshold higher than 50 were selected for data-dependent MS/MS survey scans.
10
Verapamil Transport in Microfluidic GI-Tract Model
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Verapamil was introduced into the compartmentalised GI-tract, total-analysis system at a concentration of 1 mg/mL (Fig. 1 a) in either ultrapure water or apple-juice sample matrices (1 µL/min). The final concentration of verapamil on the apical side of the flow-through transwell was 5 µg/mL. The process of automated sample clean-up and trapping was described above in the section “Compartmentalised system design and operation ”. In the case of verapamil analysis, the C8 nanotrap column was eluted towards a microfluidic chip-based iKey BEH C18 analytical column using the following gradient. The 3 µL/min microflow gradient was based on a published method23 (link) and consisted of mobile phase A (water with 1% acetonitrile) and mobile phase B (acetonitrile with 1% water), both containing 0.1% formic acid. The gradient started at 10% B and, after 4 min, was linearly increased to 100% B in 4 min. This composition was kept constant for 3 min, and then reverted to 10% B in 0.1 min. An equilibration time of 3.9 min was allowed prior to the next injection. MS detection was performed with a Waters Xevo QTOF mass spectrometer with the same settings as for the offline analysis of omeprazole. Data were collected using MassLynx, yielding a separate data file for each trap-column analysis.
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