Bile acids analysis was performed by UPLC-MS chromatography in an AQUITY I-Class (Waters Corp., Milford, MA) connected to a Xevo-G2 QTof MS detector. Separation was run on an ACQUITY UPLC BEH C18 1.7 mm column (2.1 × 100 mm, Waters Corp.) using water and acetonitrile as mobile phases, both containing a 0.1% of formic acid. Mass spectroscopy detection was performed in full-scan negative mode (100 to 1,200 Da). Concentration of bile acid was determined based on standard curves with QuanLynx software (Waters Corp.). Bile acids were extracted using the following methodologies and using chenodeoxycholic acid-d4 (CDCA-d4 ) as internal standard: lyophilized ileal digesta samples were homogenized in absence of solvent on a TissueLyzer II (QIAGEN, Hilden, Germany) and 20 mg of homogenate were extracted with 840 µL of H2 O: ACN (1:1) including internal standard, respectively. After homogenization, the mixture was centrifuged (15,000 g × 10 min, 4°C) and the supernatant diluted in H2 O: ACN (1:50) for UPLC-MS analysis. Plasma proteins were precipitated by addition of 200 µL of ACN with 5 µL internal standard to 50 µL of plasma. After centrifugation, supernatants were directly analyzed by UPLC analysis.
Acquity uplc beh c18 1.7 mm column
Manufactured by Waters Corporation
The Acquity UPLC BEH C18 1.7 mm column is an analytical column designed for use in ultra-performance liquid chromatography (UPLC) systems. It features a stationary phase of bridged ethylene hybrid (BEH) particles with a C18 bonded phase, providing high-resolution separation of a wide range of analytes.
Automatically generated - may contain errors
Lab products found in correlation
Impact hd qtof mass spectrometer, by Bruker (8 mentions)
Dionex ultimate 3000 uhplc system, by Bruker (5 mentions)
2 c18 uplc column, by Waters Corporation (3 mentions)
Poroszyme immobilized pepsin cartridge, by Thermo Fisher Scientific (2 mentions)
Ultimate 3000, by Bruker (2 mentions)
Aquity 1 class, by Waters Corporation (1 mentions)
Xevo g2 qtof ms detector, by Waters Corporation (1 mentions)
Quanlynx software, by Waters Corporation (1 mentions)
Tissuelyzer 2, by Qiagen (1 mentions)
Acquity uplc, by Waters Corporation (1 mentions)
Uplc c18 column, by Waters Corporation (1 mentions)
Ultimate 3000 uhplc system, by Bruker (1 mentions)
11 protocols using acquity uplc beh c18 1.7 mm column
1
Bile Acids Quantification by UPLC-MS
2
UPLC-ESI-QTOFMS metabolite profiling
3
Rapid Protein Digestion and Analysis
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Protein samples were rapidly thawed and injected onto an integrated fluidics system containing a HDx-3 PAL liquid handling robot and climate-controlled (2 °C) chromatography system (LEAP Technologies), a Dionex Ultimate 3000 UHPLC system, as well as an Impact HD QTOF Mass spectrometer (Bruker). The protein was run over either one (at 10 °C) or two (at 10 °C and 2 °C) immobilized pepsin columns (Trajan; ProDx protease column, 2.1 × 30 mm PDX.PP01-F32) at 200 µL/min for 3 min. The resulting peptides were collected and desalted on a C18 trap column (Acquity UPLC BEH C18 1.7 mm column (2.1 × 5 mm); Waters 186003975). The trap was subsequently eluted in line with an ACQUITY 1.7 μm particle, 100 × 1 mm2 C18 UPLC column (Waters), using a gradient of 3–35% B (Buffer A 0.1% formic acid; Buffer B 100% acetonitrile) over 11 min immediately followed by a gradient of 35–80% over 5 min. Full details of all LC methods can be found at70 (link). Mass spectrometry experiments acquired over a mass range from 150 to 2200 m/z using an electrospray ionization source operated at a temperature of 200 C and a spray voltage of 4.5 kV.
4
Rapid Protein Digestion and Mass Spectrometry
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Protein samples were rapidly thawed and injected onto an integrated fluidics system containing an HDx-3 PAL liquid handling robot and climate-controlled chromatography system (LEAP Technologies), a Dionex Ultimate 3000 ultrahigh-performance liquid chromatography system, and an Impact high-definition quadrupole orthogonal acceleration–time-of-flight mass spectrometer (Bruker). The full details of the fluidics system are described in (62 (link)). The protein was run over either one (at 10°C) or two (at 10° and 2°C) immobilized pepsin columns (Applied Biosystems Poroszyme immobilized pepsin cartridge, 2.1 mm by 30 mm; Thermo Fisher Scientific, 2-3131-00; Trajan ProDx protease column, PDX.PP01-F32, 2.1 mm by 30 mm) at 200 μl/min for 3 min. The resulting peptides were collected and desalted on a C18 trap column [Acquity UPLC BEH C18 1.7-mm column (2.1 mm by 5 mm); Waters 186003975]. The trap was subsequently eluted in line with a C18 reverse-phase separation column (Acquity 1.7-mm particle, 100 × 1 mm2 C18 UPLC column, Waters 186002352), using a gradient of 5 to 36% B (buffer A, 0.1% formic acid; buffer B, 100% acetonitrile) over 16 min. MS experiments acquired over a mass range from 150 to 2200 mass/charge ratio (m/z) using an electrospray ionization source operated at a temperature of 200°C and a spray voltage of 4.5 kV.
5
Rapid Protein Digestion and Characterization
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Protein samples were rapidly thawed and injected onto an integrated fluidics system containing a HDx-3 PAL liquid handling robot and climate-controlled (2 °C) chromatography system (LEAP Technologies), a Dionex Ultimate 3000 UHPLC system, as well as an Impact HD QTOF Mass spectrometer (Bruker). The protein was run over either one (at 10 °C) or two (at 10 °C and 2 °C) immobilized pepsin columns (Trajan; ProDx protease column, 2.1 mm x 30 mm PDX.PP01-F32) at 200 μL/min for 3 mins. The resulting peptides were collected and desalted on a C18 trap column (Acquity UPLC BEH C18 1.7 mm column (2.1 x 5 mm); Waters 186003975). The trap was subsequently eluted in line with an ACQUITY 1.7 μm particle, 100 × 1 mm2 C18 UPLC column (Waters), using a gradient of 3–35% B (Buffer A 0.1% formic acid; Buffer B 100% acetonitrile) over 11 mins immediately followed by a gradient of 35–80% over 5 mins. Mass spectrometry experiments were acquired over a mass range from 150 to 2200 m/z using an electrospray ionization source operated at a temperature of 200 °C and a spray voltage of 4.5 kV.
6
Automated Protein Complex LC-MS/MS Analysis
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Protein samples were rapidly thawed and injected onto an integrated fluidics system containing a HDx-3 PAL liquid handling robot and climate-controlled (2°C) chromatography system (Trajan), a Dionex Ultimate 3000 UHPLC system, as well as an Impact HD QTOF Mass spectrometer (Bruker). The full details of the automated LC system are described in (44 (link)). The TTC7B-FAM126A and PI4KA complex samples were run over one immobilized pepsin column (Trajan; ProDx protease column, 2.1 mm × 30 mm PDX.PP01-F32) at 200 μL/min for 3 minutes at 10°C, and the MBP-EFR3A samples were run over two immobilized pepsin columns (Waters; Enzymate Protein Pepsin Column, 300Å, 5μm, 2.1 mm × 30 mm) at 350 μL/min for 3 minutes at 2°C. The resulting peptides were collected and desalted on a C18 trap column (Acquity UPLC BEH C18 1.7mm column (2.1 mm × 5 mm); Waters 186004629). The trap was subsequently eluted in line with an ACQUITY 1.7 μm particle, 100 mm × 2.1 mm C18 UPLC column (Waters; 186003686), using a gradient of 3–35% B (Buffer A 0.1% formic acid; Buffer B 100% acetonitrile) over 11 minutes immediately followed by a gradient of 35–80% over 5 minutes. Mass spectrometry experiments acquired over a mass range from 150 to 2200 m/z using an electrospray ionization source operated at a temperature of 200°C and a spray voltage of 4.5 kV.
7
Rapid Proteome Hydrogen-Deuterium Exchange
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Protein samples were rapidly thawed and injected onto an integrated fluidics system containing a HDx-3 PAL liquid handling robot and climate-controlled chromatography system (LEAP Technologies), a Dionex Ultimate 3000 UHPLC system, as well as an Impact HD QTOF Mass spectrometer (Bruker). The protein was run over two immobilized pepsin columns (Applied Biosystems; Poroszyme™ Immobilized Pepsin Cartridge, 2.1 mm × 30 mm; Thermo-Fisher 2-3131-00; at 10°C and 2°C respectively), or for the high low human regulator HDX over one immobilized Nepenthesin-2 column from Affipro (AP-PC-004), at 200 μL/min for 3 min. The resulting peptides were collected and desalted on a C18 trap column [Acquity UPLC BEH C18 1.7 mm column (2.1 × 5 mm); Waters 186003975]. The trap was subsequently eluted in line with an ACQUITY 1.7 μm particle, 100 × 1 mm2 C18 UPLC column (Waters 186002352), using a gradient of 3–35% B (buffer A, 0.1% formic acid; buffer B, 100% acetonitrile) over 11 min immediately followed by a gradient of 35–80% B over 5 min. MS experiments acquired over a mass range from 150 to 2200 mass/charge ratio (m/z) using an electrospray ionization source operated at a temperature of 200°C and a spray voltage of 4.5 kV.
8
Rapid Protein Digestion and LC-MS Analysis
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Protein samples were rapidly thawed and injected onto an integrated fluidics system containing an HDx-3 PAL liquid handling robot and climate-controlled (2 °C) chromatography system (LEAP Technologies), a Dionex Ultimate 3000 UHPLC system, and an Impact HD QTOF mass spectrometer (Bruker). The protein was run over one immobilised pepsin column (Trajan; ProDx protease column, 2.1 mm × 30 mm PDX.PP01-F32) at 200 μl/min for 3 min at 10 °C. The resulting peptides were collected and desalted on a C18 trap column [Acquity UPLC BEH C18 1.7 mm column (2.1 × 5 mm); Waters 186003975]. The trap was subsequently eluted in line with an ACQUITY 1.7 µm particle, 100 × 1 mm2 C18 UPLC column (Waters 186002352), using a gradient of 3–35% B (buffer A, 0.1% formic acid; buffer B, 100% acetonitrile) over 11 min immediately followed by a gradient of 35–80% B over 5 min. MS experiments acquired over a mass range from 150 to 2200 mass/charge ratio (m/z) using an electrospray ionisation source operated at a temperature of 200 °C and a spray voltage of 4.5 kV.
9
Automated Proteolytic Digestion and LC-MS
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Protein samples were rapidly thawed and injected onto an integrated fluidics system containing a HDx-3 PAL liquid handling robot and climate-controlled (2 °C) chromatography system (LEAP Technologies), a Dionex Ultimate 3000 UHPLC system, as well as an Impact HD QTOF Mass spectrometer (Bruker). The full details of the automated LC system are as previously described (42 ). The protein was run over one immobilized pepsin column (Trajan; ProDx protease column, 2.1 mm × 30 mm PDX.PP01-F32) at 200 μl/min for 3 min at 8 °C. The resulting peptides were collected and desalted on a C18 trap column (Acquity UPLC BEH C18 1.7 mm column (2.1 × 5 mm); Waters; catalog no.: 186003975). The trap was subsequently eluted in line with an Acquity 1.7 μm particle, 100 × 1 mm2 C18 UPLC column (Waters), using a gradient of 3 to 35% B (buffer A 0.1% formic acid; buffer B 100% ACN) over 11 min immediately followed by a gradient of 35 to 80% over 5 min. MS experiments acquired over a mass range from 150 to 2200 m/z using an electrospray ionization source operated at a temperature of 200 °C and a spray voltage of 4.5 kV.
10
Automated Protein Digestion and LC-MS Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Protein samples were rapidly thawed and injected onto an integrated fluidics system containing a HDx-3 PAL liquid handling robot and climate-controlled (2 °C) chromatography system (LEAP Technologies), a Dionex Ultimate 3000 UHPLC system, as well as an Impact HD QTOF Mass spectrometer (Bruker). The full details of the automated LC system are described in77 (link). The protein was run over one immobilized pepsin column (Trajan; ProDx protease column, 2.1 mm × 30 mm PDX.PP01-F32) at 200 µL/min for 3 min at 10 °C. The resulting peptides were collected and desalted on a C18 trap column (Acquity UPLC BEH C18 1.7 mm column (2.1 × 5 mm); Waters 186003975). The trap was subsequently eluted in line with an ACQUITY 1.7 μm particle, 100 × 1 mm2 C18 UPLC column (Waters), using a gradient of 3–35% B (Buffer A 0.1% formic acid; Buffer B 100% acetonitrile) over 11 min immediately followed by a gradient of 35–80% over 5 min. Mass spectrometry experiments acquired over a mass range from 150 to 2200 m/z using an electrospray ionization source operated at a temperature of 200 °C and a spray voltage of 4.5 kV.
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