All reagents were commercial products and were used without further purification unless specified otherwise. 67Ga-citrate was purchased from Fujifilm RI Pharma Co., Ltd. (Tokyo, Japan). FITC was purchased from Sigma-Aldrich (St Louis, MO, USA). Fluorenylmethyloxycarbonyl (Fmoc)-ε-Ahx-OH was obtained from Merck Millipore (Billerica, MA, USA). The other Fmoc amino acids, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and hydroxybenzotriazole (HOBt), were purchased from Watanabe Chemical Industries Co., Ltd. (Hiroshima, Japan). Rink amide AM resin was purchased from Merck Millipore (Darmstadt, Germany). Mass spectra were obtained using MALDI-TOF-MS using Ultraflex MALDI TOF/TOF MS (Bruker Daltonics, Bremen, Germany). HPLC analysis was performed using a Shimadzu HPLC system (LC-10AT pump with SPD-10A UV detector; λ = 254 nm). A gamma survey meter (Aloka, Tokyo, Japan) was used as the refractive index (RI) detector. An automated gamma counter with an NaI (Tl) detector (2470 WIZARD2, Perkin-Elmer, Waltham, MA, USA) was used to measure radioactivity. A multi-mode reader (Cytation3; Biotek, Winooski, VT, USA) was used to measure absorbance and fluorescence.
Ultraflex maldi tof tof ms
Manufactured by Bruker
Sourced in Germany
The Ultraflex MALDI TOF/TOF MS is a high-performance mass spectrometry instrument designed for the analysis of biomolecules. It utilizes matrix-assisted laser desorption/ionization (MALDI) technology and time-of-flight (TOF) mass analysis to provide high-resolution and accurate mass measurements. The instrument is capable of performing both MS and MS/MS (tandem mass spectrometry) experiments, allowing for the identification and characterization of complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Lc 10at pump, by Shimadzu (2 mentions)
Cytation 3, by Agilent Technologies (2 mentions)
Hplc system, by Shimadzu (2 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
2470 wizard2, by PerkinElmer (1 mentions)
Rink amide am resin, by Merck Group (1 mentions)
Fluorescein isothiocyanate isomer 1, by Merck Group (1 mentions)
Spd 10a uv detector, by Shimadzu (1 mentions)
Scx column, by Phenomenex (1 mentions)
C18 ziptip, by Merck Group (1 mentions)
5 protocols using ultraflex maldi tof tof ms
1
Radiolabeled Peptide Probe Synthesis
2
Synthesis and Characterization of Fluorescent Peptides
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All reagents were commercial products and were used without further purification, unless otherwise indicated. Fluorescein isothiocyanate isomer I was purchased from Sigma‐Aldrich. N‐9‐fluorenylmethoxycarbonyl (Fmoc)‐ε‐Ahx‐OH was obtained from Merck Millipore. Other Fmoc amino acids, HBTU, HOBt, and Fmoc‐NH SAL resin were purchased from Watanabe Chemical Industries. Mass spectra were obtained with MALDI‐time‐of‐flight mass spectrometry (TOF‐MS) using Ultraflex MALDI‐TOF/TOFMS (Bruker Daltonics). The HPLC analysis was undertaken using a Shimadzu HPLC system (LC‐10AT pump with SPD‐10A UV detector, λ = 254 nm). A Multi‐mode Reader (Cytation3; Biotek) was used for measurement of absorbance and fluorescence.
3
Peptide Fractionation and Analysis
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The peptides containing the mixed groups were diluted with buffer A, 10 mM KH2PO4 and 25% acetonitrile, pH 3, then loaded onto a strong cationic exchange (SCX) column (Phenomenex) with 25 cm × 4.6 mm (particle size at 5 µm, 100 A). The peptides were eluted using a gradient in which buffer B (10 mM KH2PO4 and 2 M KCL in 25% ACN, pH 3.0) and a total of 39 fractions were received for future experiments. The dried peptides were examined using UltraFlex MALDI TOF/TOF MS (Bruker) to estimate the peptide content in each eluted fraction. The fractionated peptides were further pooled into seventeen groups. Subsequently, the pooled peptides were loaded onto a reversed phase column, and the separated peptides were delivered into a TripleTOF 5600 MS instrument for further identification and quantification with parallel injections.
4
Proteomic Peptide Identification Workflow
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Protein spots were manually excised from 2D-PAGE gels, and digested according to the conventional method. The dried material, including digested peptides, was dissolved in 10 μL 0.1% TFA (v/v). To remove excess salts from the extracts, solid-phase extraction was performed using a ZipTip C18 (Millipore) according to the manufacturer’s instructions. Peptides were eluted from the ZipTip with 2.5 μL 50% ACN, 0.1% TFA and 1 μL of the eluants were spotted onto a target plate. A matrix solution containing 0.3 mg/mL α-cyano-hydroxycinnamic acid in 33% acetone and 66% ethanol was prepared, and 0.5 μL of that matrix solution was immediately mixed with the sample solution in the target well. MALDI-TOF MS measurements were performed using an ultraflex MALDI-TOF/TOF-MS (Bruker Daltonics, Bremen, Germany) operating in reflector mode with 25 kV accelerating voltage and 26.5 kV reflecting voltage. MS/MS analysis was performed in LIFT mode, using post source decay. The parameters were: accelerating voltage 8.0 kV, Lift1 voltage 19.0 kV, Lift2 voltage 2.2 kV, and Reflector voltage 29.0 kV. Peptide fragments obtained by MS/MS analysis were de novo sequenced supported by DataAnalysis version 3.0 software (Bruker Daltonics).
5
MALDI-TOF Mass Spectrometry Proteomics
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The sample solutions were mixed with an equal volume of matrix solution [saturated sinapic acid or α-cyano-4-hydroxy cynnamic acid (CHCA) in 0.1% trifluoroacetic acid and 34 % acetonitrile for the specimens], and an aliquot was applied on an AnchorChip target matrix thin layer. Sinapic acid and CHCA were used for the mass spectrometric analysis of the protein species in the cell lysate and peptide fragments after trypsin-digestion, respectively.
The mass spectra were acquired in the linear positive ion mode using an Ultraflex MALDI TOF/TOF-MS (Bruker Daltonics, Inc.). Each spectrum was produced by accumulating data from 5000 consecutive laser shots. The molecular mass calibration was carried out using the #206355 Protein Calibration Standard. Both the PenSSeSPen-and NEM-reactive protein species were subjected to a database search using the Protein Information Resource (http://www-nnbrf.geogetown.edu/pirwww/). The candidate proteins were identified by the tryptic fragment mass data from the MS-Digest program of Protein Prospector (http://prospector.ucsf.edu/prospector/mshome) (Fig. S1). The heart cell lysate samples in the molecular mass range of interest were separated by dialysis using a regenerated cellulose membrane (molecular mass cutoff 6-8 kDa), then ultrafiltration through a regenerated cellulose membrane (molecular mass cutoff; 30 kDa) before the tryptic digestion.
The mass spectra were acquired in the linear positive ion mode using an Ultraflex MALDI TOF/TOF-MS (Bruker Daltonics, Inc.). Each spectrum was produced by accumulating data from 5000 consecutive laser shots. The molecular mass calibration was carried out using the #206355 Protein Calibration Standard. Both the PenSSeSPen-and NEM-reactive protein species were subjected to a database search using the Protein Information Resource (http://www-nnbrf.geogetown.edu/pirwww/). The candidate proteins were identified by the tryptic fragment mass data from the MS-Digest program of Protein Prospector (http://prospector.ucsf.edu/prospector/mshome) (Fig. S1). The heart cell lysate samples in the molecular mass range of interest were separated by dialysis using a regenerated cellulose membrane (molecular mass cutoff 6-8 kDa), then ultrafiltration through a regenerated cellulose membrane (molecular mass cutoff; 30 kDa) before the tryptic digestion.
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