The ultraperformance liquid chromatograph (UPLC) analysis was carried out, using an Acquity UPLC system (Waters Corporation, Milford, MA, USA) equipped with a binary pump, inline degasser, and autosampler coupled to an electrospray ionization quadrupole time-of-flight tandem mass spectrometer (ESI-Q-TOF/MS) (Waters Corporation, USA). Separation was carried out on BEH C18 column (100 mm × 2.1 mm, 1.7 μm, Milford, USA). The mobile phase consisted of LC grade water with 0.1% formic acid (A) and LC grade acetonitrile (B) with the following gradient profiles: 0–2 min, 5% B; 2–14 min, 5–98% B; 14–16 min, 98% B; and 16–20 min, 98–5% B. The flow rate was 0.4 mL·min−1. Before the analysis, samples were dissolved in methanol (10 mg·mL−1), centrifuged at 10,000 rpm, filtered using a 0.22 μm filter, and injected (injection volume of 15 μL).
Esi q tof ms
Manufactured by Waters Corporation
Sourced in United States
The ESI-Q-ToF-MS is a high-performance mass spectrometry instrument that combines electrospray ionization (ESI) with a quadrupole time-of-flight (Q-ToF) mass analyzer. This system is designed to provide accurate mass measurements and high-resolution analysis of a wide range of molecules.
Automatically generated - may contain errors
Lab products found in correlation
Superdex 75 10 30, by GE Healthcare (3 mentions)
Accela 600 hplc system, by Thermo Fisher Scientific (2 mentions)
Ltq orbitrap velos, by Thermo Fisher Scientific (2 mentions)
Nanoacquity uplc, by Waters Corporation (2 mentions)
Acquity uplc system, by Waters Corporation (1 mentions)
Protein a sepharose beads, by GE Healthcare (1 mentions)
Nap 5 gel filtration column, by GE Healthcare (1 mentions)
600 mhz nmr spectrometer, by Bruker (1 mentions)
Vertex 80v ftir spectrometer, by Bruker (1 mentions)
494 protein sequencer, by PerkinElmer (1 mentions)
Synapt g2 si, by Waters Corporation (1 mentions)
10 protocols using esi q tof ms
1
Quantitative LC-MS/MS Metabolite Analysis
2
Purification of Anti-HER2 Nanobodies
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Anti-HER2 nanobodies 2Rs15d, 2Rb17c and 1R136b were produced with 3 types of C-terminal amino acid tags: untagged (nanobody), His-tag (nanobody-HHHHHH), and Myc-His-tag (nanobody-AAAEQKLISEEDLNGAA-HHHHHH). Nanobodies were expressed in bacteria and purified, as described previously 22 (link). Briefly, the sequences were re-cloned into an expression vector either containing a His-tag (pHEN6), a Myc-His-tag (pHEN18), or devoid of any tag (pHEN21). The recombinant vectors were transformed into E. coli WK6 cells for nanobody expression and extraction of periplasmic proteins. His- and Myc-His-tagged nanobodies were further purified by affinity chromatography on His-Select Nickel Affinity Gel (GE Healthcare). Untagged control nanobody BcII10, recognizing a bacterial enzyme 23 (link), and both untagged 2Rb17c and 1R136b were purified on protein A Sepharose beads (GE Healthcare). The production of untagged 2Rs15d was performed according to Xavier and coworkers 16 . Final purification of all nanobodies was perfomed through size-exclusion chromatography using Superdex 75 16/60 columns (GE Healthcare) in PBS. Protein purity and integrity were evaluated using SEC on Superdex 75 10/30 (GE Healthcare) in PBS, at flow rate 0.5 mL/min. In addition, ESI-Q-ToF-MS (Waters,Micromass) was performed, in positive mode.
3
Amino Acid Sequence and MW Analysis
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The amino acid sequences of MHP6, MHP7 and MHP9 were determined using a protein sequencer (Applied Biosystems 494, Perkin Elmer Co. Ltd. Foster City, CA, USA). The MWs of MHP6, MHP7 and MHP9 were measured using an ESI-Q-TOF-MS (Micromass, Waters, Milford, MA, USA) [78 (link)].
4
Conjugation of 1B4M-DTPA to Trastuzumab
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Conjugation of 1B4M-DTPA to Trastuzumab was performed based on the protocol described by Sampath and coworkers, to yield a DTPA:Trastuzumab ratio of 5:1 34 (link). Briefly, a 100-fold molar excess of bifunctional chelator 1B4M-DTPA was conjugated overnight at RT to the free ε-amino-groups of lysines in Trastuzumab (Herceptin®, Hoffman-La Roche, Missis-sauga, ON, USA) in 3500 µl of 0.05 M sodium carbonate buffer (pH 8.5). The reaction was quenched by reducing the pH to 7.0. DTPA-Trastuzumab was purified on Superdex 75 10/30 (GE Healthcare) in 0.1 M ammonium acetate buffer pH 7.0. The degree of conjugation was evaluated with ESI-Q-ToF-MS (Waters, Micromass), in positive mode. The necessary amount of 177Lu was added to a test vial containing metal-free 0.1 M ammonium acetate buffer pH 5.0, to reach an end volume of 200 µL. Then, 100-250 µg DTPA-Trastuzumab (2.4 mg/mL) was added and incubated for 30 min at RT. 177Lu-DTPA-Trastuzumab was purified on a disposable Nap-5 gelfiltration column (GE Healthcare) and pushed through a 0.22 µm filter. Radiochemical purity was assessed using iTLC and radio- SEC, as described above.
5
Nanobody Chelator Conjugation Protocol
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A 10-fold molar excess of bifunctional chelator 1B4M-DTPA (for 177Lu) or CHX-A''-DTPA (for 111In) was conjugated for 3 h at RT to the free ε-amino-groups of lysines in the nanobodies in 600 µl of 0.05 M sodium carbonate buffer (pH 8.5). The conjugation reaction was quenched by reducing the pH of the mixture to pH 7.0. Nanobody-chelator was purified on Superdex 75 10/30 (GE Healthcare) in 0.1 M ammonium acetate buffer pH 7.0. The mean degree of conjugation was evaluated with ESI-Q-ToF-MS (Waters, Micromass), in positive mode.
6
Electrospray Ionization Mass Spectrometry and NMR Analysis
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The electrospray ionization mass spectrometry (ESI-MS) was carried out by an electrospray ionization-quadrupole-time of flight mass spectrometry (ESI-Q-TOF MS; Waters, Milford, CT, USA). The Proton Magnetic Resonance (1H-NMR) spectra were created by an AVANCE 600 MHz NMR spectrometer at a 298 K field, in the presence of tetramethylsilane as the internal standard (Bruker, Fällanden, Switzerland).
7
Comprehensive Characterization of TPB-DMTP-COF
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Fourier transformation-infrared spectroscopy (FT-IR) was performed with a Bruker Vertex 80V FT-IR spectrometer (Bruker Optics Inc., Billerica, MA. USA). Mass spectra were acquired with a nano electrospray ionization-quadrupole time-of-flight mass spectrometer (ESI Q-TOF MS) (Waters, and Milford, MA, USA) coupled with a Nano Acquity UPLC (Waters, Milford, MA, USA) or with a LTQ Orbitrap Velos coupled with an Accela 600 HPLC system (Thermo, San Jose, California). Zeta potential was measured on a Malvern ZETASIZER 2000/3000 Instrument (Malvern Instruments Ltd., Malvern Worcestershire, UK). High-resolution transmission electron microscopy (HR-TEM) images were obtained on a JEOL model JEM-2100 microscopy (JEOL, Akishima, Tokyo, Japan). BET adsorption isotherms were recorded on a Micrometrics Quadrasorb SI nitrogen adsorption–desorption apparatus (Quantachrome Instruments, Boynton Beach, FL, USA). The peptides/protein adsorption of TPB–DMTP-COF was measured on a Multiskan GO 1510 microplate reader (Thermo Fisher Scientific Inc., Vantaa, Finland).
8
Mass Spectrometry Analysis of Peptides
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The obtained peptide fractions from tryptic digests of BSA protein were analyzed with a nano electrospray ionization-quadrupole time-of-flight mass spectrometer (ESI Q-TOF MS) (Waters, and Milford, MA, USA). The samples were infused into ESI source directly with Nano Acquity UPLC (Waters, Milford, MA, USA). The full MS scan was acquired at m/z 600–1800 under positive ion mode.
The endogenous peptide fractions from human serum were identified with LTQ-Orbitrap Velos coupled with Accela 600 HPLC system (Thermo, San Jose, California). The full scan mass data were obtained from m/z 400 to 2000 (R = 60 000 at m/z 400).
The endogenous peptide fractions from human serum were identified with LTQ-Orbitrap Velos coupled with Accela 600 HPLC system (Thermo, San Jose, California). The full scan mass data were obtained from m/z 400 to 2000 (R = 60 000 at m/z 400).
9
Protein Sequence Analysis of TRP Peptides
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The sequences of TRP3, TRP6, TRP9, and TRP12 were analyzed by employing an Applied Biosystems 494 protein sequencer (Perkin Elmer, United States) (10 (link)). The MWs of TRP3, TRP6, TRP9, and TRP12 were detected by an ESI-Q-TOF-MS (Micromass, Waters, United States) (49 (link)).
10
UPLC-MS/MS Identification of Intermediates
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To confirm the nature of the intermediate identified via UPLC analysis, samples were analyzed after UPLC separation and electrospray ionization (ESI) by a quadruple time of flight MS device (ESI-Q-TOF–MS, Synapt G2-Si, Waters). Target compounds were separated using a BEH C18 column as described above. MS/MS analysis was performed at full scan mode in the m/z range of 50–1200 using an electrospray ionization (ESI) in negative mode with a capillary voltage of 1.5 kV, a source temperature of 120 °C, desolvation gas temperature of 500 °C, a gas flow of 800 L h−1 N2, and a cone gas flow of 50 L h−1 N2. Collision induced dissociation of precursor ions was performed by ramping the collision energy from 10 to 40 V. Ions with an m/z of 221.08 (± 0.01) and 165.02 (± 0.01) were monitored to identify MBP and PA, respectively. The data analysis was performed using Mass Lynx V4.1. The intermediates were identified by the mass pattern of each compound at the retention time of authentic standards.
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