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). Proteins were run over two immobilized pepsin columns (Applied Biosystems; Poroszyme Immobilized Pepsin Cartridge, 2.1 mm × 30 mm; Thermo-Fisher 2‐3131‐00; at 10 and 2°C, respectively) 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 µm column [2.1 × 5 mm]; Waters 186002350). The trap was subsequently eluted in line with a C18 reverse-phase separation column (Acquity 1.7 μm particle, 100 × 1 mm2 C18 UPLC column, Waters 186002352), using a gradient of 5–36% B (buffer A 0.1% formic acid; buffer B 100% acetonitrile) over 16 min. Lipids were directly captured on the LC system, and eluted off at the 100% acetonitrile step, with no interference on mass spectrometer or LC performance. Full details of the LC setup and gradient for lipid samples are in Stariha et al., 2021 (link). Mass spectrometry experiments were performed on an Impact II QTOF (Bruker) acquiring 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.
Impact hd qtof mass spectrometer
Manufactured by Bruker
Sourced in Germany
The Impact HD QTOF Mass Spectrometer is a high-resolution, high-mass-accuracy mass spectrometer designed for a wide range of analytical applications. It features a quadrupole time-of-flight (QTOF) mass analyzer that provides precise mass measurements and high-resolution capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Dionex ultimate 3000 uhplc system, by Bruker (9 mentions)
Acquity uplc beh c18 1.7 mm column, by Waters Corporation (8 mentions)
Ultimate 3000 uhplc system, by Bruker (4 mentions)
Acquity uplc beh c18 1.7 mm column 2 1 5 mm, by Waters Corporation (3 mentions)
2 c18 uplc column, by Waters Corporation (3 mentions)
Acquity uplc beh c18 1.7mm column 2 1 x 5 mm, by Waters Corporation (3 mentions)
Compass dataanalysis 4, by Bruker (2 mentions)
Impact 2 qtof, by Bruker (1 mentions)
Acquity uplc beh c18 1.7 m column, by Waters Corporation (1 mentions)
Tlc plate, by Merck Group (1 mentions)
Ascend 600 mhz spectrometer, by Bruker (1 mentions)
Ultimate 3000 rslc system, by Thermo Fisher Scientific (1 mentions)
Amicon ultra 15 centrifugal filter, by Merck Group (1 mentions)
Ultimate 3000 uhplc, by Thermo Fisher Scientific (1 mentions)
Avance neo, by Bruker (1 mentions)
Poroszyme immobilized pepsin cartridge, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000, by Bruker (1 mentions)
Uplc c18 column, by Waters Corporation (1 mentions)
21 protocols using impact hd qtof mass spectrometer
1
Rapid Protein Characterization by HDX-MS
2
Analytical Characterization of Organic Compounds
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The course of reactions was monitored by thin layer chromatography (TLC) using aluminum Merck TLC plates coated with silica gel 60 F254. The 1H and 13C NMR spectra were measured in a MeOD solution at 600 MHz (1H) and 151 MHz (13C) using a BRUKER ASCEND™ 600 MHZ spectrometer. Chemical shifts were reported in parts per million (ppm) with reference to the residue solvent peak for 1H and 13C (1H δH = 3.36 and 4.94 ppm, 13C δC = 47.66 ppm for MeOD). Mass spectra were obtained with HRMS (ESI) analysis using an Impact HD QTOF Mass Spectrometer (BRUKER) equipped with a Thermo Scientific/Dionex UHPLC (UltiMate 3000).
3
Rapid Protein Characterization using HDX-MS
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Protein samples for both HDX-MS and phosphorylation analysis were analyzed using the same LC-MS setup. 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) at 200 μL/min for 3 minutes. 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 minutes. 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.
4
Nano-LC-ESI-MS/MS for Peptide Identification
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Nano-LC-ESI-MS/MS was performed using an Ultimate 3000 RSLC system (Thermo Fisher Scientific) coupled to an Impact HD™ QTOF mass spectrometer (Bruker Daltonics, Bremen, Germany) via an Advance Captive Spray source (Bruker Daltonics). Peptide samples were pre-concentrated onto a C18 trapping column (THC164535, Thermo Fisher) at a flow rate of 5 μL/min in 2% (v/v) ACN 0.1% (v/v) FA for 10 minutes. Peptide separation was performed using a 75μm ID 50 cm C18 column (THC164540, Thermo Fisher) at a flow rate of 0.2 μL/minute using a linear gradient from 5 to 45% B (A: 5% (v/v) ACN 0.1% (v/v) FA, B: 80% (v/v) ACN 0.1% (v/v) FA) over 180 minutes. MS scans were acquired in the mass range of 300 to 2,200 m/z in a data-dependent fashion using Bruker’s Shotgun Instant Expertise™ method (singly charged precursor ions excluded from acquisition, CID from 23% to 65% as determined by the m/z of the precursor ion).
5
LC-MS Analysis of Chemical Compound
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LC-MS analyses were conducted by the Center for Advanced Instrumentation and the Department of Applied Chemistry at National Yang Ming Chiao Tung University, Hsinchu, Taiwan. Ultra-performance liquid chromatography (UPLC) of the sample solutions (pH 7.5) was performed via direct infusion (2 μL). High-resolution electrospray mass spectrometry (HRESI-MS) was conducted with an Impact HD Q-TOF mass spectrometer (Bruker, Karlsruhe, Germany) that was equipped with an electrospray ionization (ESI) source in positive-ion mode. The detailed ESI(+) parameters were as follows: the ion spray voltage was 4.5 kV; the capillary temperature was 200 °C; and the sheath gas flow rate was 6 L/min. The mass spectra were collected over the mass range of m/z 50–1500 at a resolving power of 40,000. The final data were analyzed using Compass DataAnalysis 4.1 (Bruker). A 20 mg/mL solution of the compound was analyzed.
6
High-throughput 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 UHPLC system, as well as an Impact HD QTOF Mass spectrometer (Bruker). The protein was run over either one immobilized pepsin column at 10 °C (Trajan; ProDx protease column, 2.1 mm × 30 mm PDX.PP01-F32) at 200 ml/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 a C18 reverse-phase separation column (Acquity 1.7 mm particle, 100 × 1 mm2 C18 UPLC column, Waters 186002352), using a gradient of 5–36% B (Buffer A 0.1% formic acid; Buffer B 100% acetonitrile) over 16 min. Full details of the LC system are described in (72 (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.
7
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.
8
Time-Resolved Analysis of PCMPS Binding to NPexo
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To track the PCMPS reaction evolution
with NPexo, we prepared the
different time-dependent binding reactions by mixing 50 μL of
500 μM purified wild-type or mutated NPexo with 5 μL of
10 mM PCMPS into 2 mL of 120 mM NaCl, 20 mM Tris-HCl, pH 7.0, 2 mM
MgCl2. The protein–ligand mixtures were then desalted
to remove the free PCMPS and salt by discontinuous diafiltration using
the Amicon Ultra-15 centrifugal filters (Millipore). The desalted
samples were dried by a vacuum concentrator and then resuspended with
50% acetonitriles and 2% formic acid for further LC-MS analysis.
An UltiMate 3000 UHPLC (Dionex) coupled with an Impact HD Q-TOF mass
spectrometer (Bruker) was used for LC-MS analysis. The chromatographic
separation was carried out on a Thermo Acclaim C18 column (2.1 ×
100 mm, 2.2 μm) with elution gradient program of 20% to 100%
acetonitrile in 20 min. The eluted samples were then protonated by
the positive ion mode of electrospray ionization source (ESI+) in
mass spectrometer. The mass spectra were collected over the mass range
of m/z 50–1500 at a resolving
power of 40 000. The collected data were processed and analyzed
by Compass Data Analysis 4.1 (Bruker, Germany). The ESI Compass Maximum
Entropy deconvolution algorithm over the mass range of 20 000
to 30 000 Da was used for neutral mass spectra analysis.
with NPexo, we prepared the
different time-dependent binding reactions by mixing 50 μL of
500 μM purified wild-type or mutated NPexo with 5 μL of
10 mM PCMPS into 2 mL of 120 mM NaCl, 20 mM Tris-HCl, pH 7.0, 2 mM
MgCl2. The protein–ligand mixtures were then desalted
to remove the free PCMPS and salt by discontinuous diafiltration using
the Amicon Ultra-15 centrifugal filters (Millipore). The desalted
samples were dried by a vacuum concentrator and then resuspended with
50% acetonitriles and 2% formic acid for further LC-MS analysis.
An UltiMate 3000 UHPLC (Dionex) coupled with an Impact HD Q-TOF mass
spectrometer (Bruker) was used for LC-MS analysis. The chromatographic
separation was carried out on a Thermo Acclaim C18 column (2.1 ×
100 mm, 2.2 μm) with elution gradient program of 20% to 100%
acetonitrile in 20 min. The eluted samples were then protonated by
the positive ion mode of electrospray ionization source (ESI+) in
mass spectrometer. The mass spectra were collected over the mass range
of m/z 50–1500 at a resolving
power of 40 000. The collected data were processed and analyzed
by Compass Data Analysis 4.1 (Bruker, Germany). The ESI Compass Maximum
Entropy deconvolution algorithm over the mass range of 20 000
to 30 000 Da was used for neutral mass spectra analysis.
9
Integrated Workflow for Protein Analysis
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Protein samples for both HDX-MS and phosphorylation analysis were analyzed using the same LC-MS setup. 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 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) 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.
10
BAFF-induced B cell proteome analysis
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Ten million B cells were treated with BAFF 1-h in the absence or presence with PP1 30-min prior to BAFF exposure. After 1-h, the extracellular medium was removed and centrifuged at 10,000 × g 15-s to pellet cells. One hundred microliter of 65% nitric acid was used to resuspend the cell pellet and this was stayed at 60 °C 3-h to ensure cell rupture and bring the cell suspension to a total volume of 5 mL by adding the distilled water. Liquid chromatography–mass spectrometry experiments were performed using an Impact HD Q-TOF mass spectrometer (Bruker, Germany), which was equipped with an electrospray ionization (ESI) source operating in positive ion mode.
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