ESI-MS experiments were performed using an Apex-Qe Ultra 7T instrument (Bruker Daltonic, Germany) equipped with an ESI source. The instrument was operated in the positive-ion mode and calibrated with the TunemixTM mixture (Bruker Daltonic, Germany). The mass accuracy was better than 5 ppm. Spectra were recorded using aqueous solutions of acetonitrile (50%) and formic acid (1%), at a protein concentration 5 μM. The sample in the high-pressure experiment was infused at a flow rate of 3 μl/min. The obtained mass spectra were analysed using a Biotools (Bruker Daltonic, Germany) software. The instrumental parameters were as follows: scan range 300–2500 m/z; drying gas nitrogen; temperature of drying gas 200°C; the potential between the spray needle and the orifice 4.5 kV; source accumulation 0.5 s; ion accumulation time 0.5 s. Analysis and deconvolution of the obtained spectra were carried out with a Biotools (Bruker) software.
Biotools
Manufactured by Bruker
Sourced in Germany, United States
BioTools is a suite of analytical instruments designed for bioanalytical applications. The core function of BioTools is to provide comprehensive solutions for the detection, identification, and characterization of biomolecules, such as proteins, peptides, and nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Flexanalysis, by Bruker (7 mentions)
Mascot, by Matrix Science (5 mentions)
Dataanalysis, by Bruker (4 mentions)
Hystar 3, by Bruker (3 mentions)
Maldi tof tof mass spectrometer, by Bruker (2 mentions)
Dataanalyis, by Bruker (2 mentions)
Snap ver 2, by Bruker (2 mentions)
Mediterranea sea c18 column, by Teknokroma (2 mentions)
Esquire 3000 ion trap, by Bruker (2 mentions)
Agilent 1100 hplc system, by Agilent Technologies (2 mentions)
Mascot software, by Matrix Science (2 mentions)
Ultraflex, by Bruker (2 mentions)
Flexanalysis software, by Bruker (2 mentions)
Hct ultra etdii ion trap mass spectrometer, by Bruker (2 mentions)
Ultimate 3000 rslcnano system, by Thermo Fisher Scientific (2 mentions)
Ultraflextreme maldi tof tof mass spectrometer, by Bruker (2 mentions)
Nanoacuity uplc, by Waters Corporation (1 mentions)
Synapt g2, by Waters Corporation (1 mentions)
Solarix, by Bruker (1 mentions)
Esquire 3000 mass spectrometer, by Bruker (1 mentions)
44 protocols using biotools
1
Structural Analysis of Proteins by ESI-MS
2
Protein Identity Confirmation by MS
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MS was used to confirm the identity of the purified protein as Sc-Rsm22. After IMAC and SEC, the solution containing the purified protein was subjected to SDS–PAGE and stained with Coomassie Blue. Protein bands were cut out of the gel, washed with water and treated with trypsin overnight. After the tryptic digestion, 2% trifluoroacetic acid was added, mixed and run on an ultrafleXtreme MALDI TOF-TOF mass spectrometer (Bruker). The data were analyzed using Bruker BioTools (Bruker) and Mascot (Matrix Science). All of the MS experiments and analysis were performed in the Biocenter Oulu Proteomics and Protein Analysis core facility.
3
Actinidin Purity Analysis via MALDI-TOF MS
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The purity of actinidin was analyzed by MALDI-TOF MS. The molecular mass of the protease, the excised spots of the SDS-PAGE was trypsinized and given incubation (37 °C) overnight. The digests were dehydrated then treated with 0.1% TCA. This mixture (0.5 ml) was suspended in 0.5 ml of cyano-4-hydroxycinnamic acid and sprayed onto the MALDI-TOF mass spectrometry target plate. The peptides and spectra were analyzed and using Bruker Auto flex (Bruker Daltonics, Germany) and Bruker BioTools version 2.2.
4
Protein Subunits Separation and Characterization
5
RP-HPLC-ESI-MS/MS Analysis of Disulfide-Linked Peptides
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For RP-HPLC analyses with UV detection (214 nm) and on-line electrospray ionization (ESI-MS/MS), an Agilent 1100 Series HPLC (Agilent Technologies, Waldbronn, Germany) and an Esquire 3000 mass spectrometer (Bruker Daltonik, Bremen, Germany) were used. To aid the identification of disulphide linked fragments, the hydrolysates were analyzed after a reducing step using dithiothreitol, at a final concentration of 70 mM and pH 7.0, for 1 h at 37°C [22 (link)].
Chromatographic separations were performed with a RP318 column (250 x 4.6 mm, Bio-Rad). The operating conditions were: flow rate, 0.8 mL/min; injection volume, 50 μl; solvent A, 0.37 mL/L trifluoroacetic acid in Milli-Q water; and solvent B, 0.27 mL/L trifluoroacetic acid in HPLC grade acetonitrile. Elution was conducted with a linear gradient of solvent B in A from 0 to 70% in 75 min, followed by 100% B for 30 min. Ion source parameters were: nebulizer pressure, 60 psi; dry gas, 12 L/min and dry temperature, 350°C. Using Data Analyses TM (version 3.0; Bruker Daltonik), the m/z spectral data were processed and transformed to spectra representing mass values. Biotools (version 2.1; Bruker Daltonik) was used to process the MS(n) spectra and to perform peptide sequencing.
Chromatographic separations were performed with a RP318 column (250 x 4.6 mm, Bio-Rad). The operating conditions were: flow rate, 0.8 mL/min; injection volume, 50 μl; solvent A, 0.37 mL/L trifluoroacetic acid in Milli-Q water; and solvent B, 0.27 mL/L trifluoroacetic acid in HPLC grade acetonitrile. Elution was conducted with a linear gradient of solvent B in A from 0 to 70% in 75 min, followed by 100% B for 30 min. Ion source parameters were: nebulizer pressure, 60 psi; dry gas, 12 L/min and dry temperature, 350°C. Using Data Analyses TM (version 3.0; Bruker Daltonik), the m/z spectral data were processed and transformed to spectra representing mass values. Biotools (version 2.1; Bruker Daltonik) was used to process the MS(n) spectra and to perform peptide sequencing.
6
Identifying CD63-binding Proteins
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COS7 cells were transfected using the CD63-HA-6× HN expression plasmid. Cell lysates were prepared from the cells at 2 days after transfection. The cell lysates were applied to Ni columns (TaKaRa). The CD63-binding proteins were eluted using imidazole-containing buffer and fractionated to obtain 0.5 ml of each. The CD63-containing fractions were subjected to SDS-PAGE and silver staining. A protein band that was detected in CD63 WT but not in the TCS mutant was picked from the SDS-PAGE gel. The gel band was destained according to the manufacturer’s instructions (Silver Stain MS kit, Wako, Japan). The protein in gel was treated with trypsin and eluted from the gel according to the manufacturer’s instructions (Bruker Daltonics, Germany). The trypsin-digested sample was mixed with the same volume of 0.7 mg/ml a-cyano-4-hydroxycinnamic acid (HCCA; Bruker Daltonics, Germany) as a matrix. The HCCA-mixed sample was analyzed by Ultraflex III matrix assisted laser desorption/ionization–time of flight mass spectrometry (Bruker Daltonics, Germany) using the associated software [flexcontrol (ver. 3.3) and flexanalysis (ver. 3.3)] and the m/z peaks were obtained. The m/z signals were analyzed using biotools (ver. 3.2; Bruker Daltonics, Germany) and matrix server (Matrix Science, United States) to identify the protein in a public database (Swiss-Prot).
7
Protein Identification by MALDI-TOF Mass Spectrometry
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As previously described [55 (link)], proteins were identified after in-gel tryptic digestion and extraction of peptides from the gel’s pieces, by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) using an AutoFlex III Smartbeam MALDI-TOF/TOF (Bruker Daltonics, Barcelona, Spain). Samples were applied to Prespotted AnchorChip plates (Bruker Daltonics, Barcelona, Spain) surrounding calibrates provided on the plates. Spectra were acquired with flexControl on reflector mode, (mass range 850–4000 m/z, reflector 1: 21.06 kV; reflector 2: 9.77 kV; ion source 1 voltage: 19 kV; ion source 2: 16.5 kV; detection gain 2.37×) with an average of 3500 added shots at a frequency of 200 Hz. Each sample was processed with flexAnalysis (version 3.0, Bruker Daltonics) considering a signal-to-noise ratio over 3, applying statistical calibration and eliminating background peaks. For identification, peaks between 850 and 1000 were not considered, as in general only matrix peaks are visible on this mass range. After processing, spectra were sent to the interface BioTools (version 3.2, Bruker Daltonics) and MASCOT search on Swiss-Prot 57.15 database was performed [Taxonomy: Homo Sapiens, Mass Tolerance 50 to 100, up to 2 miss cleavage, Global Modification: Carbamidomethyl (C), Variable Modification: Oxidation (M)]. Identification was accepted with a score higher than 56.
8
MS/MS Data Processing and Peptide Identification
9
LC-MS/MS for Amaranth Protein Identification
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LC-MS/MS analysis was performed on an Agilent 1100 HPLC System (Agilent Technologies, Waldbron, Germany) connected on-line to an Esquire 3000 ion trap (Bruker Daltonik GmbH, Bremen, Germany), and equipped with an electrospray ionization source. The reverse phase Mediterranea Sea C18 column (150×2.1 mm i.d., 5 μm particle size) (Teknokroma, Barcelona, Spain) was used. Peptides were eluted with a linear gradient of solvent B (acetonitrile:formic acid, 1000:1, v/v) in A (water:formic acid, 1000:1, v/v) going from 0% in 45% in 60 min at a flow rate of 0.2 mL/min. The injection volume was 50 L. Using Data Analysis™ (version 3.0; Bruker Daltonics), the m/z spectral data were processed and transformed to representing mass values. For peptide sequencing, the matched MS/MS spectra were interpreted by using BioTools (version 2.1; Bruker Daltonics), and MASCOT from Matrix Science (Boston, MA, USA), using a homemade database that includes the sequenced proteins of amaranth.
10
MALDI-TOF/TOF Mass Spectrometry of Peptides
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The digestion of proteins on the gels was performed as described by Shevchenko et al. (2006) , with some changes. Peptides were dissolved in 10 μL 0.1% trifluoroacetic acid. The saturated solution of 4 mg/mL alpha-cyano-4-hydroxycinnamic in 50% acetonitrile and 0.3% trifluoroacetic acid was mixed with an equal amount of sample and placed in an AnchorChip 800/384 plate (Bruker Daltonik GmbH) and dried in a laminar airflow for recrystallization. The samples were analyzed in a MALDI TOF/TOF MS (Ultraflex, Bruker Daltonik GmbH) in reflectron mode. Peptides with a signal-to-noise ratio above 100 had their MS/MS analyzed using the LIFT technology that is embedded in the Ultraflex MS; on average, five MS/MS spectra were measured for each protein digested using two to five identified peptides. The data processing was carried out using flexAnalysis and BioTools software packages (Bruker Daltonik GmbH).
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