Mass determination of prepro-DlPPO1 and pro-DlPPO1were performed by an ESI-LTQ-Orbitrap Velos (Thermo Fisher Scientific Bremen, Germany) mass spectrometer with a mass range of 200–4000 m/z and a mass accuracy close to 3 ppm with external calibration. Prior to MS 100 µg of the protein solutions were exchanged into 5 mM sodium acetate (pH 7.0) buffer. The protein solutions were diluted 100-fold in a mixture of 80%(v/v) acetonitrile and 0.1%(v/v) formic acid immediately before being applied to the mass spectrometer.
Esi ltq orbitrap velos
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The ESI-LTQ-Orbitrap Velos is a high-performance mass spectrometer that combines an electrospray ionization (ESI) source with a linear ion trap (LTQ) and an Orbitrap mass analyzer. This instrument is designed to provide high-resolution, high-mass accuracy, and sensitive detection of a wide range of analytes, making it a versatile tool for various applications in analytical chemistry and life sciences.
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Lab products found in correlation
4 protocols using esi ltq orbitrap velos
1
Mass Determination of Prepro-DlPPO1 and Pro-DlPPO1
2
Electrospray Ionization Mass Spectrometry of SIPPOs
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Electrospray Ionization Mass Spectrometry (ESI-MS) of SIPPO1 was performed on a nano electrospray ionisation–quadrupol and time-of-flight mass spectrometer (ESI-QTOF-MS, MaXis 4G UHR-TOF, Bruker) with a mass range of 50–20000 m/z applying the positive mode. Pure latent enzyme at a concentration of 10 g/l was used. The buffer was exchanged to 5 mM ammonium acetate (pH 7.0) and the enzyme solution was diluted to 1% (v/v) in 2% acetonitrile and 1‰ formic acid immediately before being applied to the mass spectrometer. Mass determination of SIPPO2 was performed by an ESI-LTQ-Orbitrap Velos (Thermo Fisher Scientific Bremen, Germany) with a mass range of 200–4000 m/z and a mass accuracy close to 3 ppm with external calibration. Prior to MS SIPPO2 solution was ultra-filtrated by centrifugation and the buffer was exchanged to 5 mM ammonium acetate (pH 7.0) and the protein solution was diluted 100 times in a mixture of 80% (v/v) acetonitrile and 0.1% (v/v) formic acid.
3
ESI-LTQ-Orbitrap Velos Mass Spectrometry
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Electrospray ionization
liquid chromatography mass spectrometry was performed in an ESI–LTQ–Orbitrap
Velos (Thermo Fisher Scientific Bremen, Germany) with a mass range
of 200–4000 m/z and a mass
accuracy close to 3 ppm with external calibration. Prior to MS measurements,
the purified PaPPO solution was ultrafiltered by
centrifugation, and the buffer system was changed to 5 mM ammonium
acetate, pH 5, in order to reduce the concentration of nonvolatile
salts to a minimum. Afterward a protein solution with a concentration
of approximately 0.53 g/L was diluted 100 times in a mixture of 80%
(v/v) acetonitrile and 0.1% (v/v) formic acid.
liquid chromatography mass spectrometry was performed in an ESI–LTQ–Orbitrap
Velos (Thermo Fisher Scientific Bremen, Germany) with a mass range
of 200–4000 m/z and a mass
accuracy close to 3 ppm with external calibration. Prior to MS measurements,
the purified PaPPO solution was ultrafiltered by
centrifugation, and the buffer system was changed to 5 mM ammonium
acetate, pH 5, in order to reduce the concentration of nonvolatile
salts to a minimum. Afterward a protein solution with a concentration
of approximately 0.53 g/L was diluted 100 times in a mixture of 80%
(v/v) acetonitrile and 0.1% (v/v) formic acid.
4
Proteomic Analysis of IFI16 Interactome
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HFF cells stably expressing either FL IFI16-eGFP, PY-eGFP, HIN-eGFP, or eGFP alone were infected with RF HSV-1 (MOI of 10). Cells were harvested 6 hpi, and immunoaffinity isolations with a GFP antibody prepared in-house were conducted as described previously (24). Eluted proteins were digested with trypsin (Promega) and analyzed by nano-liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS) with an ESI-LTQ Orbitrap Velos (Thermo Scientific). Raw MS/MS spectra were searched and extracted using Proteome Discoverer (v. 1.4), and assessed through SEQUEST HT (v. 1.4). Specificity filtering on weighted spectral counts was conducted using the SAINT (Significance Analysis of INTeractions) algorithm, and a value of 0.96 was used as a specificity threshold (see Table S1 in the supplemental material). Spectral counts were normalized by the length of the IFI16 bait (PY or HINAB) and log2 fold enrichment was assessed as PY/HIN. The specific proteins were submitted to the STRING database (45 (link)), categorized by Gene Ontology, and visualized in Cytoscape (v. 3.4.0).
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