Purified recombinant Rspo1 was separated on SDS–polyacrylamide gels. After CBB staining, the bands were excised, treated with 0.05 μg of sequencing-grade modified trypsin (Promega) and endoproteinase Asp-N (Roche Diagnostics) at 37°C for 12 h in 0.1 M Tris-HCl, pH 8.0, and reduced by propionamidation. The digests were desalted using C18μ ZipTips (Millipore) and analyzed by MALDI-TOF MS on an ultrafleXtreme TOF/TOF MS (Bruker Daltonics, Billerica, MA) in reflector mode using α-cyano-4-hydroxycinnamic acid as a matrix. The selected peaks were analyzed by MS/MS in LIFT mode.
Ultraflextreme tof tof ms
Manufactured by Bruker
Sourced in Germany
The UltrafleXtreme TOF/TOF MS is a high-performance mass spectrometry instrument manufactured by Bruker. It utilizes time-of-flight (TOF) and tandem time-of-flight (TOF/TOF) technology to provide accurate mass analysis and fragmentation capabilities for a wide range of samples.
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5 protocols using ultraflextreme tof tof ms
1
Proteomic Analysis of Recombinant Rspo1
2
MALDI-TOF Sample Preparation Protocol
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Sample preparation for characterization by MALDI involved dissolution of each sample of interest in 50%ACN/0.1% TFA at a concentration of 1 mg/ml. 2,5-Dihydroxy Benzoic acid (DHB) matrix was then prepared at a concentration of 10 mg/ml in 50%ACN/0.1%TFA. Samples were then diluted 1:1 with DHB matrix solution and spotted onto a MALDI target plate followed by measurement of m/z on a Bruker Ultra FlexTreme Tof/Tof MS.
3
Proteomic Analysis of Recombinant ECM1
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Purified recombinant ECM1 was subjected to SDS–polyacrylamide gels. After CBB staining, the bands were excised with trypsin (TPCK-treated, Worthington Biochem. Co.). The digests were desalted using ZipTip C18μ tips (EMD Millipore Co., Billerica, MA) and applied to MALDI-TOF MS on an ultrafleXtreme TOF/TOF MS (Bruker Daltonics, Bremen, Germany) in reflector mode using α-cyano-4-hydroxycinnamic acid as the matrix. The selected peaks were analyzed by MS/MS in LIFT mode.
4
Proteome Analysis by Mass Spectrometry
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Proteome analysis was performed to identify the proteins. Protein bands were excised from SDS-10%PAGE gels after CBB staining. To remove the dye from the gel, a de-staining solution (30% acetonitrile, 50 mM NH4HCO3) was added and incubated for 30 min. Then, 60% acetonitrile and 20 mM NH4HCO3 were added to remove water from the gel. Next, 5% (w/w) trypsin (Promega Japan, Tokyo, Japan) was added to the dried gel and incubated at 37 °C for 12 h. Peptides released from the gel were analysed by mass spectrometry using an UltrafleXtreme TOF/TOF MS (Bruker Daltonics GmbH, Bremen, Germany) operating in positive reflection ion mode between m/z 0 and 5000 Da.
5
MALDI-TOF/TOF Analysis of Pin1 Modifications
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An Autoflex
Speed TOF MS or an Ultraflextreme TOF/TOF MS (Bruker
Daltonics), both equipped with a Nd:YAG (solid state) laser operating
at 355 nm, were used to obtain spectra. All spectra were obtained
in positive ion mode. Peptide-CHCA solutions (1 μL) were deposited
on 384-spot MALDI target plates and air-dried prior to analysis. Full
mass spectra of peptides were obtained in reflectron mode on the Ultraflextreme,
using a 500–4500 mass range. Spectra from treated and untreated
samples were overlaid to identify peaks corresponding to masses appearing
in spectra from ONE-treated Pin1 samples which did not appear in unmodified
Pin1 samples. Selected peptide ions were dissociated using LIFT on
the TOF/TOF. TOF/TOF fragmentation data were interrogated using FlexAnalysis
software and analyzed against a theoretical Pin1 peptide digest using
Protein Prospector.
Speed TOF MS or an Ultraflextreme TOF/TOF MS (Bruker
Daltonics), both equipped with a Nd:YAG (solid state) laser operating
at 355 nm, were used to obtain spectra. All spectra were obtained
in positive ion mode. Peptide-CHCA solutions (1 μL) were deposited
on 384-spot MALDI target plates and air-dried prior to analysis. Full
mass spectra of peptides were obtained in reflectron mode on the Ultraflextreme,
using a 500–4500 mass range. Spectra from treated and untreated
samples were overlaid to identify peaks corresponding to masses appearing
in spectra from ONE-treated Pin1 samples which did not appear in unmodified
Pin1 samples. Selected peptide ions were dissociated using LIFT on
the TOF/TOF. TOF/TOF fragmentation data were interrogated using FlexAnalysis
software and analyzed against a theoretical Pin1 peptide digest using
Protein Prospector.
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