Standard glycopeptide analyses were performed on a Bruker Ultraflex III MALDI-TOF/TOF MS (Bruker, Daltonics, Germany). One microliter of sample was dropped on the MALDI plate and dried at room temperature. Then, 1 μL of DHB matrix solution (20 mg/mL, ACN/H2O/TFA, 60:39.9:0.1, v/v/v) was spotted on the MALDI plate and air dried. For HeLa exosome proteins, the LC-MS/MS analyses was carried out using an easy nLC-1000 system coupled with a Fusion Lumos mass spectrometer (Thermo Fisher Scientific, USA) using an ESI nanospray source. Mobile phase A was composed of 0.1% FA in water, and 0.1% FA in ACN was prepared as mobile phase B. The total flow rate was 600 nL/min, and the gradient was performed as follows: 6% to 9% buffer B for 8 min; 9% to 14% buffer B for 16 min; 14% to 30% buffer B for 36 min; 30% to 40% buffer B for 15 min; and 40% to 95% buffer B for 3 min. After eluting with 95% buffer for 7 min, the separation system was equilibrated with 6% buffer B for 5 min. The spray voltage was operated at 2.3 kV. The MS/MS spectra were acquired in data-dependent acquisition mode, and the full mass scan was acquired for m/z from 300 to 1,400 with a resolution of 120,000.
Ultraflex 3 maldi tof tof ms
Manufactured by Bruker
Sourced in Germany
The Ultraflex III MALDI-TOF/TOF MS is a mass spectrometry system designed for sensitive and high-resolution analysis of biomolecules. It utilizes matrix-assisted laser desorption/ionization (MALDI) technology combined with time-of-flight (TOF) and tandem time-of-flight (TOF/TOF) mass analyzers to provide accurate mass measurements and structural information.
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7 protocols using ultraflex 3 maldi tof tof ms
1
Glycopeptide and Exosome Proteome Analysis
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MALDI-TOF/TOF MS Analysis of Single Cells
3
MALDI-TOF/TOF MS Protein Identification Protocol
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In positive ion reflector mode, an Ultraflex III MALDI-TOF/TOF MS (Bruker, Karlsruhe, Baden- Württemberg, Germany) was used to perform MS analysis. Four of the most intense ion signals were automatically chosen as precursors for MS/MS acquisition, excluding the matrix ion signals and trypsin autolysis peaks. MASCOT Version 2.2 (Matrix Science, London, UK) and Biotools software (Version 3.2, Bruker) were used to search the MS/MS spectra combined peptide mass fingerprint in the NCBInr database. The following search criteria were used: Homo sapiens, trypsin cleavage, oxidation of methionines allowed as variable modification, one missed cleavage allowed, carbamidomethylation as fias modification, peptide mass tolerance at 75 ppm, and fragment tolerance at 0.5 Da. A successfully identified protein had the following parameters: ion score confidence interval (CI%) for PMF and MS/MS data ⩾95%, distinct sequences identified in MS/MS analysis ⩾2 and peptide count (hit) ⩾4.
4
MALDI-TOF/TOF MS Analysis of Single Cells
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A Bruker Ultraflex III MALDI-TOF/TOF MS (Bruker Daltonics) equipped with a Smartbeam laser and LIFT-TM cell was used to obtain MS and MS/MS spectra with Bruker Compass v. 1.2 software. Because the cells contain relatively small quantities of peptide, all spectra were obtained from 50 laser shots per acquisition. MS spectra were obtained in positive ion reflectron mode, with an m/z range of 500–4000 Da. Instrument settings were optimized for maximum detection sensitivity using the Bruker Flex Control software: ion source 1 voltage, 25.0 kV; ion source 2 voltage, 20.5 kV; reflector 1 voltage, 26.5 kV; reflector 2 voltage, 14.5 kV; lens voltage, 9.5 kV. Synthetic peptide standards were not suitable for calibration of the instrument because the laser intensity needed for analysis of single cells was often far too high when analyzing pure standard, making it impossible to identify the monoisotopic peak of the ion needed for accurate calibration. Therefore, a reference spectrum from a freshly dissected A. suum AVK neuron, with known peptide peaks [23 (link)], was used for external calibration of the instrument.
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Peptide Identification via MALDI-TOF/TOF MS
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The peptide mixtures were identified on a Bruker Ultraflex III MALDI-TOF/TOF MS (Bruker Daltonics, Germany) operating in reflectron mode with 20 kV accelerating voltage and 23 kV reflecting voltage. Peptide mass fingerprints (PMFs) were searched against the SwissProt database using the program Mascot 2.1 (Matrix Science Ltd). The search parameters were as follows: trypsin digestion with one missed cleavage; carbamidomethyl modification of cysteine as a fixed modification; oxidation of methionine as a variable modification; peptide tolerance maximum of ±0.5 Da; MS/MS tolerance maximum of ±100 ppm; peptide charge of +1; and p < 0.05 for a local PMF search.
6
MALDI-MSI Protein Analysis Protocol
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In both experiments, a standard sample processing and analysis pipeline was applied for MALDI-MSI of proteins, using a Bruker Ultraflex III MALDI-TOF/TOF MS (Bruker Daltonics).
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MALDI-TOF/TOF Mass Spectrometry Protocol
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MALDI-TOF/TOF mass spectrometry (MS) measurements were performed on a Bruker Ultraflex III MALDI-TOF/TOF MS (Bruker Daltonics, Billerica, MA, USA) operating in reflectron mode. A saturated solution of a-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.1% trifluoroacetic acid was used as the matrix. The SNAP algorithm in FlexAnalysis 3.4 was used to identify the 150 most prominent peaks. The subsequent tandem MS (MS/MS) analysis was performed in a data-dependent manner, and the five most abundant ions were subjected to high-energy, collisioninduced dissociation analysis. The collision energy was set to 1 keV; nitrogen was used as the collision gas.
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