To assess the mass of the monomeric form, intact mass measurements were performed under denaturing conditions by injecting the samples into Acquity UPLC Protein column BEH C4 (300 Å, 1.7 µm, 1 × 150 mm, Waters, Milford, MA, U.S.A.) using a Dionex Ultimate 3000 analytical RSLC system (Dionex, Germering, Germany) coupled to a HESI source (Thermo Fisher Scientific, Bremen, Germany). The separation was performed with a flow rate of 90 µl/min by applying a gradient of solvent B from 15 to 20 % in 2 min, then from 20 to 45 % within 10 min, followed by column washing and re-equilibration steps. Solvent A was composed of MilliQ water with 0.1% formic acid, while solvent B consisted of acetonitrile with 0.1% formic acid. Eluting fractions were analysed on high resolution QExactive HF-HT-Orbitrap-FT-MS benchtop instrument (Thermo Fisher Scientific, Bremen, Germany). Protein mode was activated with resolution of 15 000, in-source CID of 25 eV, AGC target of 3e6 and averaging 10 µscans. The scan range was set to 600–2000 m/z. Data analysis was performed with Protein Deconvolution 4.0 (Thermo Fischer Scientific, Sunnyvale, CA, USA) using Respect algorithm.
Acquity uplc protein beh c4 column
Manufactured by Waters Corporation
Sourced in United States
The ACQUITY UPLC Protein BEH C4 Column is a high-performance liquid chromatography (HPLC) column specifically designed for the separation and analysis of proteins. The column features a bonded C4 stationary phase and is compatible with ultra-high-pressure liquid chromatography (UPLC) systems. This column is suitable for a variety of protein analyses, including protein characterization, purification, and peptide mapping.
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Lab products found in correlation
Dionex ultimate 3000, by Thermo Fisher Scientific (2 mentions)
Xevo g2 xs qtof analyzer, by Waters Corporation (2 mentions)
H class instrument, by Waters Corporation (2 mentions)
Unifi software, by Waters Corporation (2 mentions)
Acquity uplc system, by Waters Corporation (2 mentions)
Acquity classic uplc, by Waters Corporation (2 mentions)
Xevo g2 xs, by Waters Corporation (2 mentions)
Qexactive hf ht orbitrap ft ms benchtop instrument, by Thermo Fisher Scientific (1 mentions)
Protein deconvolution 4, by Thermo Fisher Scientific (1 mentions)
Thermo orbitrap elite, by Thermo Fisher Scientific (1 mentions)
Acquity uplc 1 class system, by Waters Corporation (1 mentions)
Protein deconvolution 2, by Thermo Fisher Scientific (1 mentions)
Masslynx mass spectrometry software 4, by Waters Corporation (1 mentions)
Acquity uplc 1 class plus, by Waters Corporation (1 mentions)
Xevo g2 xs qtof ms system, by Waters Corporation (1 mentions)
Unifi 1, by Waters Corporation (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
Zorbax 300sb c8 column, by Agilent Technologies (1 mentions)
1290 infinity 2 uplc, by Agilent Technologies (1 mentions)
Bioconfirm software, by Agilent Technologies (1 mentions)
20 protocols using acquity uplc protein beh c4 column
1
Monomeric Mass Determination by LC-MS
2
Protein Mass Analysis by UPLC-MS
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To assess the mass of the monomeric form, intact mass measurements were performed under denaturing conditions by injecting the samples into Acquity UPLC Protein column BEH C4 (300 Å, 1.7 µm, 1 x 150 mm, Waters, Milford, MA, U.S.A.) using a Dionex Ultimate 3000 analytical RSLC system (Dionex, Germering, Germany) coupled to a HESI source (Thermo Fisher Scienti c, Bremen, Germany). The separation was performed with a ow rate of 90 µl/min by applying a gradient of solvent B from 15 to 20% in 2 min, then from 20 to 45% within 10 min, followed by column washing and re-equilibration steps. Solvent A was composed of MilliQ water with 0.1% formic acid, while solvent B consisted of acetonitrile with 0.1% formic acid. Eluting fractions were analyzed on high resolution QExactive HF-HT-Orbitrap-FT-MS benchtop instrument (Thermo Fisher Scienti c, Bremen, Germany). Protein mode was activated with resolution of 15 000, in-source CID of 25 eV, AGC target of 3e6 and averaging 10 µscans. The scan range was set to 600-2000 m/z. Data analysis was performed with Protein Deconvolution 4.0 (Thermo Fischer Scienti c, Sunnyvale, CA, USA) using Respect algorithm.
3
Intact Protein Mass Measurements of SUMO-sfGFP
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Intact protein mass measurements of purified SUMO-sfGFP variants were performed by electrospray LC-MS on a Waters H-class instrument with a Waters Acquity UPLC protein BEH C4 column (300 Å, 1.7 μm, 2.1 mm × 50 mm). The following gradient used a flow rate of 0.3 mL/min: A: 0.01% formic acid in H2O; B: 0.01% formic acid in MeCN. 5–95% B 0–6 min. Mass analysis was conducted with a Waters Xevo G2-XS QTof analyzer. Proteins were ionized in positive ion mode applying a cone voltage of 40 kV. Raw data were analyzed employing the maximum entropy deconvolution algorithm. The data were exported and plotted with QtiPlot (version 0.9.9.7).
4
Intact Protein Mass Analysis by UPLC-MS
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The intact masses of the proteins were analyzed using a Waters ACQUITY I class UPLC system (Milford, MA) with an ACQUITY UPLC Protein BEH C4 column (2.1 mm × 100 mm, 1.7 μm particle size; Waters). The mobile phases were 0.1% formic acid in water (eluent A) and 0.1% formic acid in acetonitrile (eluent B). The gradient applied was: 0–3 min, 5% eluent B; 3–13 min, linear increase to 50% eluent B at 0.2 ml/min. The eluent was injected into a Thermo Orbitrap Elite (Thermo Fisher Scientific, Waltham, MA) and ionized with an electrospray source. MS spectra were acquired in the mass range of 400–2,000 m/z and 120,000 resolution at m/z 200. The deconvoluted mass spectra were generated using Protein Deconvolution 2.0 (Thermo Fisher Scientific, Waltham, MA).
5
UCHL1 Inhibitor Screening by Mass Spectrometry
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Samples
of 1.4 μM UCHL1 in 70 μL buffer containing 50
mM Tris·HCl, 100 mM NaCl at pH 7.6, 2.0 mM cysteine, and 1 mg/mL
3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic
acid (CHAPS) were prepared. These samples were treated with 1 μL
of DMSO or 1 μL of a 10 mM inhibitor/probe stock solution in
DMSO (140 μM final concentration) and incubated for 30 min at
room temperature. Samples were then diluted 3-fold with water and
analyzed by mass spectrometry by injecting 1 μL into a Waters
XEVO-G2 XS Q-TOF mass spectrometer equipped with an electrospray ion
source in positive mode (capillary voltage 1.2 kV, desolvation gas
flow 900 L/h, T = 60 °C) with a resolution of R = 26 000. Samples were run using two mobile phases:
(A) 0.1% formic acid in water and (B) 0.1% formic acid in CH3CN on a Waters Acquity UPLC protein BEH C4 column [300 Å, 1.7
μm (2.1 × 50 mm2), flow rate = 0.5 mL/min, run
time = 14.00 min, column T = 60 °C, and mass
detection 200–2500 Da]. Gradient: 2–100% B. Data processing
was performed using Waters MassLynx mass spectrometry software 4.1,
and ion peaks were deconvoluted using the built-in MaxEnt1 function.
of 1.4 μM UCHL1 in 70 μL buffer containing 50
mM Tris·HCl, 100 mM NaCl at pH 7.6, 2.0 mM cysteine, and 1 mg/mL
3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic
acid (CHAPS) were prepared. These samples were treated with 1 μL
of DMSO or 1 μL of a 10 mM inhibitor/probe stock solution in
DMSO (140 μM final concentration) and incubated for 30 min at
room temperature. Samples were then diluted 3-fold with water and
analyzed by mass spectrometry by injecting 1 μL into a Waters
XEVO-G2 XS Q-TOF mass spectrometer equipped with an electrospray ion
source in positive mode (capillary voltage 1.2 kV, desolvation gas
flow 900 L/h, T = 60 °C) with a resolution of R = 26 000. Samples were run using two mobile phases:
(A) 0.1% formic acid in water and (B) 0.1% formic acid in CH3CN on a Waters Acquity UPLC protein BEH C4 column [300 Å, 1.7
μm (2.1 × 50 mm2), flow rate = 0.5 mL/min, run
time = 14.00 min, column T = 60 °C, and mass
detection 200–2500 Da]. Gradient: 2–100% B. Data processing
was performed using Waters MassLynx mass spectrometry software 4.1,
and ion peaks were deconvoluted using the built-in MaxEnt1 function.
6
Proteomic Analysis of Histone Methyltransferase
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The purified proteins after histone methyltransferase reaction were analyzed on an Xevo G2-XS QTOF MS System (Waters Corporation) equipped with an electrospray ionization (ESI) source in conjunction with Waters ACQUITY UPLC I-Class plus. Separation and desalting were carried out on a Waters ACQUITY UPLC Protein BEH C4 Column (300 Å, 2.1 × 50 mm, 1.7 μm). Mobile phase A was 0.1% formic acid in water and mobile phase B was acetonitrile with 0.1% formic acid. A constant flow rate of 0.2 ml/min was used. Data was analyzed using Waters UNIFI software. Mass spectral deconvolution was performed using UNIFI software (version 1.9.4, Waters Corporation).
7
Characterization of EGFRvIII-BsAb Protein
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The purified EGFRvIII-BsAb was characterized with LC/MS, SEC-HPLC, and other methods. The molecular weight of the produced EGFRvIII-BsAb was determined with an LC/MS method. A sample of 0.5–1 mg was concentrated to 10 mg/mL and dialyzed into N-glycan excision buffer (5 mM NH4HCO3, 40867; Fluka, Charlotte, N.C., USA) and mixed with 1 μL PNGase F (500 U/μL, P0704S; New England Biolab, Ipswich, MA, USA) at 37 ℃ for 24 h to remove the N-glycan. Then, the components in the sample were separated with the ACQUITY UPLC Protein BEH C4 Column (186004495; Waters, Etten-Leur, Netherlands). A Waters Acquity VION IMS Q-Tof mass spectrometer (Milford, MA, USA) was coupled with the UPLC (Milford, MA, USA) to determine the mass of the target protein. The data were collected and processed with UNIFI 1.8 software (Waters, Milford, MA, USA). Purity of the EGFRvIII-BsAb was determined by the SEC-HPLC method (Agilent, Santa Clara, CA, USA).
8
Evaluation of Protein C4 Columns
9
Mass Spectrometric Analysis of OTUB2 Inhibition
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Samples of 1.4 μM OTUB2 in 70 μL of
buffer containing
50 mM Tris·HCl, 100 mM NaCl at pH 7.6, and 2.0 mM cysteine were
prepared. These samples were treated with 1 μL of DMSO or 1
μL of a 10 mMLN5P45 stock solution in DMSO (140
μM final concentration) and incubated for 30 min at room temperature.
Samples were then diluted 3-fold with water and analyzed by mass spectrometry
by injecting 1 μL into a Waters XEVO-G2 XS Q-TOF mass spectrometer
equipped with an electrospray ion source in positive mode (capillary
voltage 1.2 kV, desolvation gas flow 900 L/h, T =
60 °C) with a resolution of R = 26,000. Samples
were run using two mobile phases: (A) 0.1% formic acid in water and
(B) 0.1% formic acid in CH3CN on a Waters Acquity UPLC
protein BEH C4 column [300 Å, 1.7 μm (2.1 × 50 mm2), flow rate = 0.5 mL/min, run time = 14.00 min, column T = 60 °C, and mass detection 200–2500 Da].
Gradient: 2–100% B. Data processing was performed using Waters
MassLynx mass spectrometry software 4.1, and ion peaks were deconvoluted
using the built-in MaxEnt1 function.
buffer containing
50 mM Tris·HCl, 100 mM NaCl at pH 7.6, and 2.0 mM cysteine were
prepared. These samples were treated with 1 μL of DMSO or 1
μL of a 10 mM
μM final concentration) and incubated for 30 min at room temperature.
Samples were then diluted 3-fold with water and analyzed by mass spectrometry
by injecting 1 μL into a Waters XEVO-G2 XS Q-TOF mass spectrometer
equipped with an electrospray ion source in positive mode (capillary
voltage 1.2 kV, desolvation gas flow 900 L/h, T =
60 °C) with a resolution of R = 26,000. Samples
were run using two mobile phases: (A) 0.1% formic acid in water and
(B) 0.1% formic acid in CH3CN on a Waters Acquity UPLC
protein BEH C4 column [300 Å, 1.7 μm (2.1 × 50 mm2), flow rate = 0.5 mL/min, run time = 14.00 min, column T = 60 °C, and mass detection 200–2500 Da].
Gradient: 2–100% B. Data processing was performed using Waters
MassLynx mass spectrometry software 4.1, and ion peaks were deconvoluted
using the built-in MaxEnt1 function.
10
SARS-CoV-2 3CL Protease Molecular Weight Analysis
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Molecular weight of SARS-CoV-2 3CLpro with or without compounds was analyzed through Agilent 1290 Infinity II UPLC and Q-TOF 6530 mass spectrometry. The detailed experimental procedure was performed as follows. 8.4 μM SARS-CoV-2 3CLpro with 90 μM Vitamin K3 or 5,8-dihydroxy-1,4-naphthoquinone was incubated in reaction buffer (0.1 M PBS, 1 mM EDTA, pH 7.4) for 30 min at 37 °C. 10 μL protein samples were separated on a Waters ACQUITY UPLC Protein BEH C4 column (2.1 × 50 mm, 1.7 μm, 300 Å). The mobile phase A is 0.1% formic acid in ddH2O and mobile phase B is 0.1% formic acid in acetonitrile. The column temperature is 60 °C and the flow rate is 0.3 mL/min. The gradient is 10% MPB (mobile phase B) from 0 to 1 min, then 10% to 50% MPB from 1 min to 6 min, 50% to 90% MPB from 6 min to 7.5 min and keep 90% MPB for 0.5 min. The mass spectrometry parameters were shown as follows. Positive scan mode on ESI source; Scan mass range: 100–3200 m/z; Gas temperature: 300 °C; Drying gas: 8 L/min; Nebulizer: 35 psi; Sheath gas temperature: 350 °C; Drying gas: 8 L/min; Nebulizer: 35 psi; Sheath gas temperature: 350 °C; Sheath gas flow: 12 L/min. The acquired data is processed with Agilent BioConfirm software (version 10.0).
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