Urine samples were diluted in formic acid (0.1% (v/v) in HPLC grade water) to a protein concentration of approximately 5 pmol/μl. The samples were injected onto a C4 desalting trap (Waters MassPREP™ Micro desalting column, 2.1 × 5 mm, 20 μm particle size, 1000 Å pore size) (Waters, Manchester, UK) that was fitted on a Waters nano ACQUITY Ultra Performance liquid chromatography® (UPLC®) system. The chromatography system was coupled to a Waters SYNAPT™ G1 QTof mass spectrometer fitted with an electrospray source. Protein was eluted over a 10 min acetonitrile (ACN) gradient (5–95% (v/v)) at 40 μl/min. Data were collected between 500–3500 m/z. The data were processed using maximum entropy deconvolution (MAX ENT 1, Mass Lynx version 4.1, Waters) at 0.5 Da/channel over a mass range of 8500–10000 Da. The mass spectrometer was calibrated externally with horse heart myoglobin (1 pmol/μl, Sigma).
Synapt g1 q tof mass spectrometer
Manufactured by Waters Corporation
Sourced in United States
The Synapt G1 Q-TOF mass spectrometer is an analytical instrument designed for high-performance mass analysis. It utilizes quadrupole time-of-flight (Q-TOF) technology to provide accurate and sensitive detection of various molecular compounds. The core function of the Synapt G1 is to separate and detect ions based on their mass-to-charge ratio, enabling the identification and characterization of complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Horse heart myoglobin, by Merck Group (1 mentions)
Massprep micro desalting column, by Waters Corporation (1 mentions)
Max ent 1 mass lynx version 4, by Waters Corporation (1 mentions)
Nano acquity ultra performance liquid chromatography, by Waters Corporation (1 mentions)
Amicon centrifugal filter, by Merck Group (1 mentions)
Acquity uplc, by Waters Corporation (1 mentions)
Beh c18, by Waters Corporation (1 mentions)
Chenodeoxycholic acid, by Merck Group (1 mentions)
Cholic acid, by Merck Group (1 mentions)
Tauro α muricholic acid, by Steraloids (1 mentions)
Aquity uplc system, by Waters Corporation (1 mentions)
Deoxycholic acid, by Merck Group (1 mentions)
Mouse total bile acids assay kit, by Crystal Chem (1 mentions)
4 protocols using synapt g1 q tof mass spectrometer
1
Urine Protein Analysis by LCMS
2
Protein Extraction and Identification from Titanium
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Proteins were extracted from titanium discs. Briefly, three titanium disks from each condition were incubated with 5 mL of DMEM supplemented with 10% bovine serum for 3 h at 37°C in 5% CO2, rinsed with PBS twice and incubated at room temperature for 20 min with 1 mL of extraction buffer solution (8 M urea, 10 mM DTT, 100 mM Tris pH 8.6). Solution was removed with the aid of a cell scraper, pooled, washed with 50 mM NH4CO3 and concentrated to a final volume of 50 µL using a 3 kDa cutoff Amicon centrifugal filter (Millipore). Samples were then prepared and digested with trypsin and identified by LC/MS in a nanoacquity nanoUPLC system coupled to a Synapt G1 Q-TOF mass spectrometer (Waters, USA). Data was processed for identification and quantification of proteins using Proteinlynx Global Server v2.5.
3
Purification and Analysis of Compounds
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Compounds were purified using a Biotage Isolera One system with a 12-g SiliCycle C18 17% cartridge, 230- to 400-μm particle size, and a 40- to 63-μm mesh or the Shimadzu LC-20AT high pressure liquid chromatograph with a Restek Ultra II C18 reversed-phase column (150 mm length, 4.6-mm i.d., 5.0-μm particle size, and 100-Å pore size). Liquid-chromatography/mass spectrometry was performed on a Waters Synapt G1 Q-TOF mass spectrometer coupled to a Waters Acquity UPLC. Data were routinely collected under positive electrospray conditions.
4
Quantification of Plasma Bile Acids
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Total bile acids from plasma and gallbladder bile were measured with Mouse Total Bile Acids Assay Kit (Crystal Chem, Zaandam, Netherlands) according to the manufacturer’s protocol.
Individual bile acids were measured from plasma. To remove plasma proteins, 50 µl of EDTA-plasma was mixed with 500 µl of acetonitrile, kept for 1 h at −20 °C and centrifuged. The supernatant was dried in a vacuum and redissolved into 50 µl of 50% acetonitrile. 5 µl of the solution was analyzed by LC/MS using Aquity UPLC system (Waters, Milford, MA, USA) connected to Synapt G1 Q-TOF mass spectrometer (Waters). The eluents were A: 5 mM ammoniumacetate, 0.018% formic acid and B: 5% acetonitrile in methanol. The linear gradient was operated at 0.4 ml/min with the following program: 0 min 40% B; 10 min 100% B, 14 min 100% B, 15 min 40% B. The column, BEH C18, 1.7 µm, 2 × 100 mM (Waters), was kept at 40 °C. The mass spectrometer collected 0,2 second scans in the mass range between 50–1500 in V-mode using negative ionization, recording centroid peaks with lock mass (Leu-encephalin) correction. Quantification was calibrated with standards at 1, 5, 10, 30, 70 and 100 ng/ml. Standards were cholic acid (C1129; Sigma), chenodeoxycholic acid (C9377, Sigma), deoxycholic acid (D2510, Sigma) and tauro α-muricholic acid (C1893–000, Steraloids Inc., Newport, RI, USA).
Individual bile acids were measured from plasma. To remove plasma proteins, 50 µl of EDTA-plasma was mixed with 500 µl of acetonitrile, kept for 1 h at −20 °C and centrifuged. The supernatant was dried in a vacuum and redissolved into 50 µl of 50% acetonitrile. 5 µl of the solution was analyzed by LC/MS using Aquity UPLC system (Waters, Milford, MA, USA) connected to Synapt G1 Q-TOF mass spectrometer (Waters). The eluents were A: 5 mM ammoniumacetate, 0.018% formic acid and B: 5% acetonitrile in methanol. The linear gradient was operated at 0.4 ml/min with the following program: 0 min 40% B; 10 min 100% B, 14 min 100% B, 15 min 40% B. The column, BEH C18, 1.7 µm, 2 × 100 mM (Waters), was kept at 40 °C. The mass spectrometer collected 0,2 second scans in the mass range between 50–1500 in V-mode using negative ionization, recording centroid peaks with lock mass (Leu-encephalin) correction. Quantification was calibrated with standards at 1, 5, 10, 30, 70 and 100 ng/ml. Standards were cholic acid (C1129; Sigma), chenodeoxycholic acid (C9377, Sigma), deoxycholic acid (D2510, Sigma) and tauro α-muricholic acid (C1893–000, Steraloids Inc., Newport, RI, USA).
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