LC-MS/MS analysis of tryptic peptides mixtures was performed using a Waters SYNAPT™ HDMS™ system (Waters Corp., Milford, MA, USA). The 1D-nanoscale LC was constructed with a NanoAcquity UPLC system (Waters Corp., Milford, MA, USA). In total, 4 µL of digested peptides were injected onto the reversed-phase analytical column (20 cm × 75 μm) packed with a 1.7 μm Bridged Ethyl Hybrid (BEH) C18 material (Waters Corp., Milford, MA, USA). Peptides were eluted with a linear gradient from 2% to 40% acetonitrile developed over 60 min at a flow rate of 350 nL/min. This was followed by a 15 min period of 80% acetonitrile to clean the column before returning to 2% acetonitrile for the next sample. The effluent samples were electrosprayed into a mass spectrometer (Synapt HDMS) for MS/MS analysis of peptides. Argon gas was used in the collision cell to obtain MS/MS data. MS/MS spectra obtained were processed using Maxent 3, a deconvolution software for peptides (Ensemble 1, Iterations 50, auto peak width determination) within MassLynx 4.0. The experiment was performed in 5 replicates.
Synapt hdms system
Manufactured by Waters Corporation
Sourced in United States
The Synapt HDMS system is a high-resolution mass spectrometry instrument designed for advanced analytical applications. It employs a hybrid quadrupole-ion mobility-time of flight (Q-IM-TOF) architecture to provide high-performance separation and detection of complex samples. The system is capable of performing accurate mass measurements, ion mobility separation, and tandem mass spectrometry (MS/MS) analysis.
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Lab products found in correlation
Nanoacquity uplc system, by Waters Corporation (2 mentions)
Beh c18 material, by Waters Corporation (2 mentions)
Ftir 8400s spectrophotometer, by Shimadzu (1 mentions)
Perkin elmer 241 polarimeter, by PerkinElmer (1 mentions)
Pack ods a column, by YMC (1 mentions)
Uv 2501pc, by Shimadzu (1 mentions)
Waters 2489 uv, by Waters Corporation (1 mentions)
Beh c18 column, by Waters Corporation (1 mentions)
Sigmaplot v11, by Grafiti LLC (1 mentions)
Hplc system, by Shimadzu (1 mentions)
Kieselgel 60 f254, by Merck Group (1 mentions)
Avance 3, by Bruker (1 mentions)
Ft ir 6100 spectrometer, by Jasco (1 mentions)
P 1010 polarimeter, by Jasco (1 mentions)
Acquity beh c18 column, by Waters Corporation (1 mentions)
Acquity uplc, by Waters Corporation (1 mentions)
Beh300 c18 column, by Waters Corporation (1 mentions)
Masslynx 4, by Waters Corporation (1 mentions)
Nanoacquity system, by Waters Corporation (1 mentions)
Agilent 1200 lc system, by Agilent Technologies (1 mentions)
14 protocols using synapt hdms system
1
LC-MS/MS Analysis of Tryptic Peptides
2
Comprehensive Analytical Characterization
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Optical rotations were measured on a Perkin-Elmer 241 polarimeter (Perkin-Elmer, Waltham, MA, USA), and UV spectra were recorded on Shimadzu UV-2501 PC (Shimadzu, Kyoto, Japan). IR data were recorded using a Shimadzu FTIR-8400S spectrophotometer (Shimadzu, Kyoto, Japan). 1H and 13C NMR data were acquired using Bruker 500 and Bruker 600 instruments (Bruker, Rheinstetten, Germany), with solvent signals (CD3OD: δH 3.30/δC 49.0 ppm;) as references. HRESIMS data were acquired using a Q-TOF analyzer in SYNAPT HDMS system (Waters, Milford, MA, USA). X-ray diffraction data were collected on the Agilent GEMINITME instrument (CrysAlisPro software, Version 1.171.35.11; Agilent, Santa Clara, CA, USA). HPLC was performed using Waters 2535 system (Waters, Milford, MA, USA), with the following components: preparative column, a Daisogel-C18-100A (10 μm, 30 × 250 mm, ChuangXinTongHeng Sci. & Tech., Beijing, China) and a YMC-Pack ODS-A column (5 μm, 10 × 250 mm, YMC, Kyoto, Japan); and detector, Waters 2489 UV. Sephadex LH-20 (40–70 μm, Pharmacia Biotech AB, Uppsala, Sweden), silica gel (60–100, 100–200 and 200–300 mesh) and silica gel GF254 sheets (0.20–0.25 mm) (Qingdao Marine Chemical Plant, Qingdao, China) were used for column chromatography and TLC, respectively. TLC spots were visualized under UV light and by dipping into 5% H2SO4 in EtOH, followed by heating.
3
UHPLC-MS Analysis of Metabolites
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MS analysis was performed using a Waters Synapt HDMS system (Waters). Analytical separation was performed on a BEH C18 column (2.1 mm × 100 mm, i.d.; 1.7 μm, Waters) at 30 °C using mobile phases A (water/formic acid, 99.9: 0.1, v/v) and B (acetonitrile) at a flow rate of 0.5 mL/min. The gradient elution procedure was conducted with 7% (v/v) B for 0–2 min, 7% to 25% (v/v) B for 2–8 min, and 60% (v/v) B for 9 min.
4
Proteomic Profiling of Extracellular Vesicles
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Lysates of MVs isolated from E14tg2a.4 ES cells (30 μg) were resolved by SDS–PAGE and then stained with a Colloidal Blue Staining kit. The proteins were excised from the gel and trypsin-digested. Cornell's Proteomics Facility analysed the resulting peptide fragments using a triple quadrupole linear ion trap (4000 Q Trap) online LC/MS/MS system (Applied Biosystems/MDS Sciex) or the Synapt HDMS system (Waters). Proteins were identified by performing peptide alignment searches using the NCBI mouse RefSeq protein database.
5
Electrospray Ionization Mass Spectrometry for Kinetic Studies
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ESI MS measurements were carried out on a Synapt HDMS system (Waters Corp., UK) adapted for high-mass measurements.[26 (link)] Typical settings were: capillary voltage, 3.5 kV; cone voltage, 70 V; extraction cone voltage 2 V; trap collision energy, 5 V; transfer collision energy 6 V; bias 40 V and backing pressure, 2.0–2.8 mbar. All ESI-MS experiments were carried out at RT.
Reactions were initiated by mixing the two solutions at the outlet of the inner capillary. The data was obtained for 20 different time points, ranging from 1.06 s to 28.29 s. Spectra were recorded using the spectral mode by monitoring the entire mass spectrum of the reaction at fixed distances between the inner and outer capillaries.[11 (link)] For every time point, three independent experiments were acquired, in which 180 s acquisition time was summed and analyzed without smoothing.
The calculated reaction time is the sum of the dead time and increasing mixing volume as a function of the solutions linear velocity at a fixed volume for each time point. Relative abundances were assessed from the peak heights of the species being monitored. Line-fitting was achieved using Sigmaplot v11.0.0.77 by Systat Software Inc. (San Jose, CA, USA) and error bars represent standard deviations from the mean.
Reactions were initiated by mixing the two solutions at the outlet of the inner capillary. The data was obtained for 20 different time points, ranging from 1.06 s to 28.29 s. Spectra were recorded using the spectral mode by monitoring the entire mass spectrum of the reaction at fixed distances between the inner and outer capillaries.[11 (link)] For every time point, three independent experiments were acquired, in which 180 s acquisition time was summed and analyzed without smoothing.
The calculated reaction time is the sum of the dead time and increasing mixing volume as a function of the solutions linear velocity at a fixed volume for each time point. Relative abundances were assessed from the peak heights of the species being monitored. Line-fitting was achieved using Sigmaplot v11.0.0.77 by Systat Software Inc. (San Jose, CA, USA) and error bars represent standard deviations from the mean.
6
Characterization of Purified SCI-57 Protein
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A 30 µM purified SCI-57 sample obtained from A2–A7 fraction concentration and solvent exchange into formic acid 0.1% by 3 kDa filter was analyzed using the SYNAPT-HDMS system (Waters Corp., USA) with ESI-Lockspray interphase. Prior to the experiments, the system was calibrated with a sodium iodide standard solution (Waters Corp., USA), and the European Pharmacopoeia Reference standard insulin (EP insulin) sample was analyzed as a positive control (30 µM).
In the experiment, data was acquired by injecting the protein solution directly into the interphase. We used a scan interval of 50–2,000 m/z with an ionizing spray voltage of 3.2 kV in positive ionization mode and a desolvation temperature of 110°C to acquire data. Molecular weight estimations were generated by the UniDec GUI versión 1.1.10 (Marty et al., 2015 (link)).
In the experiment, data was acquired by injecting the protein solution directly into the interphase. We used a scan interval of 50–2,000 m/z with an ionizing spray voltage of 3.2 kV in positive ionization mode and a desolvation temperature of 110°C to acquire data. Molecular weight estimations were generated by the UniDec GUI versión 1.1.10 (Marty et al., 2015 (link)).
7
Characterization of Organic Compounds
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Optical rotation was measured using a P-1010 polarimeter (Jasco) in chloroform at 27 °C. IR spectra were recorded on a FT/IR-6100 spectrometer (Jasco). NMR spectra were recorded on a 500 MHz NMR AVANCE III (Bruker) using deuterated chloroform (CDCl3) and deuterated benzene (C6D6). MS spectra were obtained using a SYNAPT HDMS system (Waters). Preparative TLC was performed using silica gel plates (Merck Kieselgel 60 F254). Silica gel (Kanto Chemical, Silica gel 60 N, spherical, neutral, 100–210 µm) was used for column chromatography. Semi-preparative HPLC was performed on a Shimadzu HPLC system with a Cosmosil πNAP (10 × 250 mm) column.
8
Proteome Analysis by LC-MS/MS
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LC-MS/MS analysis of digested peptide mixtures was performed using a Waters SYNAPT™ HDMS™ system. The 1D-nanoLC was carried out with a Waters nano ACQUITY UPLC system. Four microliters of digests was injected onto the RP analytical column (20 cm × 75 μm) packed with a 1.7 μm Bridged Ethyl Hybrid (BEH) C18 material (Waters). Tryptic peptides were eluted with a linear gradient from 2% to 40% acetonitrile developed over 60 min at a flow rate of 350 mL/min. This was followed by a 15 min period of 80% acetonitrile to clean the column before returning to 2% acetonitrile for the next sample. The effluent samples were electrosprayed into a mass spectrometer (Synapt HDMS) for MS/MS analysis of peptides. Argon gas was used in the collision cell to obtain MS/MS data. MS/MS spectra thus obtained were processed using Max Ent 3, deconvolution software for peptides (Ensemble 1, Iterations 50, auto peak width determination) within Mass Lynx 4.0. The protein spectral data used in this study have been deposited at ProteomeXchange: PXD030242 and JPST001417.
9
High-Resolution Mass Spectrometry Protocol
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A Waters SYNAPT™ HDMS™ system (Waters Corp., Milford, USA). equipped with an electrospray ionization (ESI) source and hybrid Q/TOF-MS was utilized. The full-scan MS data with accurate mass measurement was acquired at positive and negative ion mode from 50 Da to 1000 Da with a 0.3 s scan time, respectively. The optimal capillary voltage was set at 3000 V for ESI+ and 2600 V for ESI−, and the sample cone voltage at 40 V, the extraction cone voltage at 4 V. Nitrogen was used as the dry gas; the desolvation gas flow rate was set at 500 L/h, and the con gas flow rate was maintained at 50 L/h. The desolvation temperature was set at 350°C and the source temperature at 110°C. All the data were acquired using an independent reference lock mass via the LockSpray™ interface to ensure accuracy and reproducibility during the MS analysis. Leucine enkephalin was used as the reference compound ([M + H]+ =556.2771 at the positive ion mode and [M − H]− =554.2615 at the negative ion mode) at a concentration of 0.2 ng/mL under a flow rate of 100 μL/min. The data were collected in the centroid mode, and the LockSpray frequency set at 10 s and averaged over 10 scans for the correction.
10
HPLC-MS Analysis of Bioactive Compounds
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The most active fraction obtained from the RP-HPLC was analyzed by Waters Synapt HDMS system (Waters, Milford, MA, USA) on an RP-C18 column (150 × 0.15 mm) with a flow rate of 0.3 mL/min. The column temperature was 30 °C and the detection wavelength was 220 nm. The mobile phase consisted of acetonitrile with 0.1% formic acid (mobile phase A) and 0.1% formic acid aqueous solution (mobile phase B). The following linear gradient was used: 0–50 min, 4% A; 50–54 min, 50% A; 54–60 min, 100% A. For mass spectrometry, parameters were set as follows: collision energy, 6 eV; ion source temperature, 100 °C; detector voltage, 1800 V; mass range, 300–2000 m/z.
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