A Thermo Scientific Q-Exactive HF-X hybrid Quadrupole-Orbitrap mass spectrometer was used for the high-resolution ESI-MS spectra and CID-MS/MS analysis of paenithopeptins using electrospray ionization in the positive ion mode. Liquid chromatography was performed on a Thermo Vanquish HPLC interfaced to the aforementioned mass spectrometer. A Thermo Scientific ProSwift RP-4H reversed phased monolith column with dimensions 1 × 250 mm was used for the separation. Solvent A was 0.1% formic acid in water and Solvent B was 0.1% formic acid in acetonitrile, with the flow rate being 200 μL/min. The LC gradient used started at 10% B for 1 min then increased to 100% B over 10 min where it remained for 5 min. MS1 scans were obtained in the orbitrap analyzer which scanned from 500–2000 m/z at a resolution of 60,000 (at 200 m/z). For collision-induced dissociation tandem mass spectrometry (CID-MS/MS), the relevant parent ion was selected with a 2 m/z window and fragmented by CID, using normalized collision energies of 20, 25 & 30 eV (results were combined into one spectrum). Fragment ions were then sent to the Orbitrap for mass analysis at 30,000 resolution. The MS data was analyzed by Thermo Xcalibur (4.2.47).
Proswift rp 4h
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The ProSwift RP-4H is a reversed-phase high-performance liquid chromatography (HPLC) column. It is designed for the separation and purification of various biomolecules, such as peptides, proteins, and oligonucleotides. The column features a monolithic silica-based stationary phase, which provides high permeability and fast mass transfer, enabling efficient and high-resolution separations.
Automatically generated - may contain errors
Lab products found in correlation
Xcalibur, by Thermo Fisher Scientific (1 mentions)
Q exactive hf x hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Vanquish hplc, by Thermo Fisher Scientific (1 mentions)
Impact 2 q tof mass spectrometer, by Bruker (1 mentions)
Q exactive focus, by Thermo Fisher Scientific (1 mentions)
4 protocols using proswift rp 4h
1
Paenithopeptin Mass Spectrometry Analysis
2
Mass Spectrometric Analysis of Deglycosylated EPO
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EPO was denatured, reduced, alkylated and digested by PNGase F as
mentioned above. de-N-Glycosylated EPO samples were injected into
a LC system (Waters ACQUITY, Milford, MA, USA), coupled with a hybrid
quadrupole-orbitrap mass spectrometer. Solvent A was 0.1% FA in water,
and solvent B was 0.1% FA in ACN. Using a RP monolithic column (ProSwift
RP-4H, 1 × 250 mm, Thermo Fisher Scientific, Bremen, Germany),
solvent B started with 5% for a duration of 2 min, increasing to 25%
in 7 min, and to 60% in 34 min at a flow rate of 0.2 mL/min. Detailed
MS instrument parameters are described in theSupporting Information .
To deconvolute the MS raw data,
the parameters of BioPharma Finder were set as follows: the amino
acid sequence of epoetin beta was used as a FASTA database; the oxidation
of methionine and carbamidomethylation of cysteine residues were included
as variable modifications; the deamidation on Asn-24, Asn-38, and
Asn-83 was set as a static modification. For the processing method,
the m/z range was set from 1500 to 3000; Xtract (isotopically resolved)
was used as a deconvolution algorithm; the output mass range was set
from 16,000 to 30,000; charge states between 8 and 25 were considered,
and a minimum number of 6 was used for deconvolution; the mass tolerance
was set to 20 p.p.m..
mentioned above. de-N-Glycosylated EPO samples were injected into
a LC system (Waters ACQUITY, Milford, MA, USA), coupled with a hybrid
quadrupole-orbitrap mass spectrometer. Solvent A was 0.1% FA in water,
and solvent B was 0.1% FA in ACN. Using a RP monolithic column (ProSwift
RP-4H, 1 × 250 mm, Thermo Fisher Scientific, Bremen, Germany),
solvent B started with 5% for a duration of 2 min, increasing to 25%
in 7 min, and to 60% in 34 min at a flow rate of 0.2 mL/min. Detailed
MS instrument parameters are described in the
To deconvolute the MS raw data,
the parameters of BioPharma Finder were set as follows: the amino
acid sequence of epoetin beta was used as a FASTA database; the oxidation
of methionine and carbamidomethylation of cysteine residues were included
as variable modifications; the deamidation on Asn-24, Asn-38, and
Asn-83 was set as a static modification. For the processing method,
the m/z range was set from 1500 to 3000; Xtract (isotopically resolved)
was used as a deconvolution algorithm; the output mass range was set
from 16,000 to 30,000; charge states between 8 and 25 were considered,
and a minimum number of 6 was used for deconvolution; the mass tolerance
was set to 20 p.p.m..
3
Phosphorylation Status Verification of Recombinant p-MLKL
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To verify the phosphorylation status of recombinant p-MLKL pseudokinase domain, the purified proteins were separated by reverse-phase chromatography on a 25 cm ProSwift RP-4H monolith column (Thermo Scientific) using a micro-flow HPLC (M-class, Waters). The HPLC was coupled to an Impact II QTOF mass spectrometer (Bruker) equipped with an ESI source. Proteins were loaded directly onto the column for online buffer exchange at a constant flow rate of 30 μL/min with buffer A (99.9% Milli-Q water, 0.1% formic acid (FA)) using a trap-valve to direct flow to waste. After 10 min, the trap valve was switched to direct flow to the mass spectrometer, and proteins were eluted with a 15-min linear gradient from 2 to 90% buffer B (99.9% acetonitrile (ACN), 0.1% FA). The Impact II QTOF was operated in mode using Compass Hystar 5.1. Settings for the 40 samples per day method were as follows: Mass Range 100 to 3000 m/z, Capillary Voltage 4500 V, Dry Gas 4 L/min, Dry Temp 200 °C.
4
Quantitative Proteomics of Synthetic Peptides
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Each peptide was analyzed by LC-MS using a Q Exactive Focus mass spectrometer (Thermo Fisher Scientific). Peptide separation was performed on Ultimate 3000 nanoHPLC and Vanquish ultra-high-performance liquid chromatography systems. The C1, C1MC1, and C2MC2 peptides were analyzed using a Proswift RP4H (Thermo Fisher) monolithic nanocolumn (0.1 × 250 mm), and the C2MC1 and C1MC2 peptides were analyzed using a Zorbax Eclipse Plus C18 Rapid Resolution HT column (2 × 50 mm, 1.8 μm, 95 Å). Acetonitrile gradients between 10%–30% and 15%–25% in formic acid (0.1%) were used. Mass analysis was performed at a resolution of 35,000 (m/z, 200) with a MS range of 500–1300 and MS/MS analysis. The collision energy was optimized for each peptide (between 22% and 25%) to reduce formation of internal fragments. The lock mass option was activated to enhance mass accuracy. For each peptide, several scans (between 5 and 10) were merged to enhance the quality of the data. Data were deconvoluted using Xtract tools included in the Freestyle software suite, version 1.3 (Thermo Electron). All daughter ions observed were verified and annotated manually.
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