The protein solution at 1.2 μg/μl was diluted in water to 24 ng/μl. Ten μl was loaded (~240 ng) onto a Waters NanoAcquity LC with online desalting. Reversed phase separation was carried out on an in‐house packed C2 column (100 μm i.d., ~50 cm long, packing material SMTC2MEB2–3‐300 from Separation Methods Technologies, Newark, DE). Mobile phases were 0.2% formic acid in water (A) and 0.2% formic acid in acetonitrile (B). A linear gradient with a flow rate of 0.3 μl/min was run from 15% to 50% mobile phase B over 30 min. The protein eluted between 30%–40% mobile phase B. Mass spectrometry data were collected on a Thermo Orbitrap Exploris. The mass spectrum (120 k resolution, 5 microscans) across the elution window were summed, and deconvoluted with FreeStyle (v1.5, ThermoScientific) for estimating the relative abundance of the phosphorylated protein.
Freestyle v1
Manufactured by Thermo Fisher Scientific
Sourced in United States
The FreeStyle v1.5 is a piece of laboratory equipment manufactured by Thermo Fisher Scientific. It is a versatile instrument designed for various scientific applications. The core function of the FreeStyle v1.5 is to perform tasks related to laboratory workflows and processes.
Automatically generated - may contain errors
Lab products found in correlation
Orbitrap exploris 480, by Thermo Fisher Scientific (2 mentions)
Amicon unltra 0.5 ml centrifugal filters, by Merck Group (2 mentions)
Nanoacquity lc, by Waters Corporation (1 mentions)
Orbitrap exploris, by Thermo Fisher Scientific (1 mentions)
Q exactive plus, by Thermo Fisher Scientific (1 mentions)
Orbitrap fusion tribrid, by Thermo Fisher Scientific (1 mentions)
Uhplc system, by Agilent Technologies (1 mentions)
7 protocols using freestyle v1
1
Phosphoprotein Quantification via LC-MS
2
Determining His-rMdumPLA2 Intact Mass
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Intact mass of the His-rMdumPLA2 was determined by direct infusion ESI-MS in a Q-Exactive Plus® instrument (Thermo Scientific, Waltham, MA, USA). Proteins were dissolved in 50% acetonitrile/water containing 0.1% formic acid at 50–100 μg/mL and infused at 5 μL/min into a HESI source, to acquire a full MS scan in the 800–2000 m/z range, at resolution of 140,000 (at m/z 240), in positive mode. The acquired MS spectra of the multiply charged ion series were deconvoluted using Freestyle® v.1.5 (Thermo, Scientific, Waltham, MA, USA) to obtain monoisotopic masses [60 (link)].
3
Carbamylation of M1-linear diubiquitin
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Carbamylation of M1-linear diubiquitin, purified as described earlier, was carried out by incubation of the dimer in 0.1 M potassium cyanate (AK Scientific) in 1X PBS pH 7.4 at 37°C for 24 h. The carbamylated M1-linear diubiquitin was buffer exchanged by size exclusion chromatography the next day into 1X PBS. Carbamylation was assessed by direct infusion on an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific). The protein solution was desalted using Amicon Unltra 0.5 mL centrifugal filters (MWCO 3 kDa, Millipore). Then the diluted protein solution at ~ 0.1 μM in 50% acetonitrile 0.1% formic acid was infused at 3 μL/min with HESI source at sheath gas 5, auxiliary gas 7, spray voltage 3.2 kV, ion transfer tube at 320°C, and funnel RF level at 50%. Spectra were acquired at 240k resolution setting in positive mode, and deconvoluted by Xtract within FreeStyle v1.5 (Thermo Fisher Scientific).
4
Carbamylation of M1-Linked Diubiquitin
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Carbamylation of M1-linear diubiquitin, purified as described earlier, was carried out by incubation of the dimer in 0.1 M potassium cyanate (AK Scientific) in 1X PBS pH 7.4 at 37˚C for 24 h. The carbamylated M1-linear diubiquitin was buffer exchanged by size exclusion chromatography the next day into 1X PBS. Carbamylation was assessed by direct infusion on an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific). The protein solution was desalted using Amicon Unltra 0.5 mL centrifugal filters (MWCO 3 kDa, Millipore). Then the diluted protein solution at ~ 0.1 µM in 50% acetonitrile 0.1% formic acid was infused at 3 µL/min with HESI source at sheath gas 5, auxiliary gas 7, spray voltage 3.2 kV, ion transfer tube at 320 °C, and funnel RF level at 50%. Spectra were acquired at 240k resolution setting in positive mode, and deconvoluted by Xtract within FreeStyle v1.5 (Thermo Fisher Scientific).
5
Intact Mass Spectrometry of Proteins
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Proteins were adjusted to a final concentration of 6 µM in 0.1% (v/v) formic acid (FA) for HPLC-MS analysis for untreated samples. For reduced samples, DTT was added to the protein to a final concentration of 10 mM. Proteins were incubated at 60°C for 30 min and adjusted to 6 µM in 0.1% (v/v) FA. Intact mass spectrometry on all proteins was carried out as described previously (Yu et al., 2021 (link)). Data were analysed using the Free Style v.1.4 (Thermo Fisher Scientific) protein reconstruct tool across a mass range of m/z 500–2000 and compared against the theoretical (sequence-based) monoisotopic mass.
6
High-Throughput Protein Analysis by HPLC-MS
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Proteins were adjusted to 10 μM in 0.1% (vol/vol) formic acid (FA) for high-pressure liquid chromatography (HPLC)-MS analysis. The samples were then injected onto an Agilent UHPLC system. Each sample was first desalted for 2 min on an Agilent (Santa Clara, CA, U.S.A.) C3 trap column (ZORBAX StableBond C3) at a flow rate of 500 μl/min at 95% buffer A (0.1% FA, vol/vol) and 5% buffer B (0.1% FA and 100% acetonitrile) followed by separation over 8 min using a 5 to 80% (vol/vol) gradient of buffer B at a flow rate of 500 μl/min. Eluted material was analyzed using a Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, Waltham, MA, U.S.A.). MS acquisition was performed using the Intact Protein Mode script. The acquisition was performed across m/z 200 to 4,000 with an accumulation time of 1 s. Data were analyzed using the Free Style v.1.4 (Thermo Fisher Scientific) protein reconstruct tool across a mass range of m/z 500 to 2,000. The expected sizes of the proteins were searched.
7
HPLC-MS Analysis of Reduced Proteins
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For untreated samples, proteins were adjusted to a final concentration of 6 µM in 0.1% (v/v) formic acid (FA) for HPLC-MS analysis. For reduced samples, DTT was added to the protein to a final concentration of 10 mM. Proteins were incubated at 60°C for 30 minutes and adjusted to 6 µM in 0.1% (v/v) FA. Intact mass spectrometry on all proteins was carried out as described previously [34] . Data were analysed using the Free Style v.1.4 (Thermo Fisher Scientific) protein reconstruct tool across a mass range of m/z 500 -2000 and compared against the theoretical (sequence based) monoisotopic mass.
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