For N-glycan analysis, 1–2 μg of purified BmVAL-1 was reduced and denatured for 10 min at 95 °C in PBS containing 1.3% (w/v) SDS and 0.1% (v/v) β-mercaptoethanol. SDS was neutralized by adding 2% (v/v) NP-40 prior to overnight digestion at 37 °C with trypsin (Sigma-Aldrich, USA), immobilized to N-hydroxysuccinimide-activated sepharose (GE Healthcare). trypsin beads were removed from the digestion mix by centrifugation and the pH of the mix was adjusted to 5 using 1 M sodium acetate. PNGase A (0.5 mU; Roche, Switzerland) was used to release N-glycans from BmVAL-1 while incubating overnight at 37 °C. The incubation mixture was applied to C18 Bakerbond™ SPE cartridges (JT Baker, USA) and the N-glycans were extracted from the flow-through on Extract Clean™ Carbo SPE columns. Eluted N-glycans were labeled with anthranilic acid (Sigma-Aldrich) and desalted by hydrophilic interaction chromatography on Biogel P10 (BioRad, USA). Samples in 75% acetonitrile were mixed with 1 μl of matrix solution (20 mg/ml 2,5-dihydroxybenzoic acid in 50% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid) and were dried under a stream of warm air. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectra (MS) were obtained using an Ultraflex II mass spectrometer (Bruker Daltonics, USA).
Ultraflex 2 mass spectrometer
Manufactured by Bruker
Sourced in United States, Germany
The Ultraflex II is a high-performance mass spectrometer designed for advanced analytical applications. It features a high-resolution time-of-flight (TOF) analyzer that enables precise mass measurements. The core function of the Ultraflex II is to provide accurate and sensitive analysis of a wide range of samples.
Automatically generated - may contain errors
Lab products found in correlation
Anthranilic acid, by Merck Group (4 mentions)
Biogel p10, by Bio-Rad (4 mentions)
Pngase a, by Roche (4 mentions)
Trypsin, by Merck Group (4 mentions)
C18 bakerbond spe cartridges, by Avantor (4 mentions)
N hydroxysuccinimide activated sepharose, by GE Healthcare (3 mentions)
Extract clean carbo spe columns, by Avantor (3 mentions)
Esi calibrant solution, by Agilent Technologies (2 mentions)
Microtof 2 instrument, by Bruker (2 mentions)
C18 column, by Silicycle (1 mentions)
Flexanalysis 2, by Bruker (1 mentions)
13 protocols using ultraflex 2 mass spectrometer
1
BmVAL-1 N-Glycan Analysis Protocol
2
N-Glycan Analysis of Purified BmVAL-1
3
Metabolite Extraction and Antimicrobial Analysis
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Metabolite extraction and antimicrobial activity analysis for LC-MS was conducted as previously described by Choi et al. (2008 (link)). The final methanol extract was evaporated and dissolved in 2 ml water. The concentrated samples were further purified on a C18 column (Sili-Cycle Inc. Quebeck, CA) and eluted with a water/methanol gradient. Active eluted fractions were used for LC-MS. LC-MS was performed using a high pressure liquid chromatography system (Thermo Electron Co, USA). The samples were injected into reverse phase columns and analyzed in a mixed solvent of water and acetonitrile containing 0.1% formic acid (0.2 ml/min).
Lipopeptide extraction for MALDI-TOF MS was performed as previously described by Li et al. (2007 (link)). Bruker Ultraflex II mass spectrometer using alpha-hydroxycinnamic acid (alpha-HCCA) was used as matrix and spectra were visualized with the mMass software (Niedermeyer and Strohalm, 2012 (link)).
Lipopeptide extraction for MALDI-TOF MS was performed as previously described by Li et al. (2007 (link)). Bruker Ultraflex II mass spectrometer using alpha-hydroxycinnamic acid (alpha-HCCA) was used as matrix and spectra were visualized with the mMass software (Niedermeyer and Strohalm, 2012 (link)).
4
Characterization of BlpLc Hydrolysis Products
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The site specificity of the BlpLc was determined using commercially available native proteins, namely myoglobin, hemoglobin, ovalbumin, and ovamucoid. The hydrolysis products were identified by MALDI-TOF and tandem MALDI-TOF/TOF using a Bruker Ultraflex II mass spectrometer (Bruker, Bremen, Germany).
Hydrolysis was conducted in a 100-mM ammonium bicarbonate buffer, pH 7.8, or in a 5-mM Britton–Robinson buffer, pH from 8.8 to 9.2. The reaction mixture contained 56 μg/mL enzyme and 400 μg/mL substrate. The mixture was incubated for 5, 15, 30, 60, and 120 min at 37 °C. The spectra were recorded using 2.5-dihydroxybenzoic acid (DHB).
Hydrolysis was conducted in a 100-mM ammonium bicarbonate buffer, pH 7.8, or in a 5-mM Britton–Robinson buffer, pH from 8.8 to 9.2. The reaction mixture contained 56 μg/mL enzyme and 400 μg/mL substrate. The mixture was incubated for 5, 15, 30, 60, and 120 min at 37 °C. The spectra were recorded using 2.5-dihydroxybenzoic acid (DHB).
5
Mass Spectrometric Analysis of PA-dPEG24
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To analyze the oligomeric state of PA-dPEG24, matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) was performed on peptide in 0.1% formic acid on an Ultraflex II mass spectrometer (Bruker Daltonic, Billerica, MA) with measurements in both the reflectron and linear mode using α-cyano 4-hydroxycinnamic matrix. The reflectron mode has a higher resolution compared to the linear mode, but only measures effectively up to 4500Da, whereas the linear mode method has a higher mass range with measurements up to 20kDa. Thus, as the predicted mass of PA-dPEG24 is 2773 Da, to detect any larger peptide oligomers, both the reflectron and linear mode were utilized.
6
N-Glycan Analysis of HpVAL-4 Protein
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For N-glycan analysis, 1–2 μg of purified HpVAL-4 was reduced and denatured for 10 min at 95 °C in PBS containing 1.3% (w/v) SDS and 0.1% (v/v) β-mercaptoethanol. SDS was neutralised by adding 2% (v/v) NP-40 prior to overnight digestion at 37 °C with trypsin (Sigma–Aldrich, USA) immobilised to N-hydroxysuccinimide-activated Sepharose (GE Healthcare). trypsin beads were removed from the digestion mix by centrifugation and the pH of the mix was adjusted to 5 using 1 M sodium acetate. PNGase A (0.5 mU; Roche, Switzerland) was used to release N-glycans from HpVAL-4 while incubating overnight at 37 °C. The incubation mixture was applied to C18 Bakerbond™ SPE cartridges (JT Baker, USA) and the N-glycans were extracted from the flow-through on Extract Clean™ Carbo SPE columns. Eluted N-glycans were labelled with anthranilic acid (Sigma–Aldrich) and desalted by hydrophilic interaction chromatography on Biogel P10 (BioRad). Samples in 75% acetonitrile were mixed with 1 μl of matrix solution (20 mg/ml of 2,5-dihydroxybenzoic acid in 50% acetonitrile, 0.1% (v/v) trifluoroacetic) and were dried under a stream of warm air. Matrix-assisted laser desorption/ionisation (MALDI) time-of-flight mass spectra (MS) were obtained using an Ultraflex II mass spectrometer (Bruker Daltonics, USA).
7
N-Glycan Analysis of Purified HpVAL-4
8
High-Resolution Mass Spectrometry of Oligosaccharides
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High-resolution electrospray ionization mass spectrometry was performed in the negative ion mode using a micrOTOF II instrument (Bruker, Germany). Oligosaccharide samples (~50 ng · L−1) were dissolved in a 1:1 (vol/vol) water-acetonitrile mixture and injected with a syringe at a flow rate of 3 μL · min−1. The capillary entrance voltage was set at 3,200 V, and the interface temperature was 180°C. Nitrogen was used as a drying gas. The mass range was from m/z 50 to m/z 3,500. Internal calibration was done with the ESI Calibrant Solution (Agilent Technologies, USA).
MALDI-TOF mass spectra were registered on a Bruker Ultraflex II mass spectrometer that was equipped with the panoramic delay of ion extraction electronics with the registration of positive ions in a linear mode using an accelerating voltage of 20 kV. 2,5-dihydroxybenzoic acid was used as a matrix.
MALDI-TOF mass spectra were registered on a Bruker Ultraflex II mass spectrometer that was equipped with the panoramic delay of ion extraction electronics with the registration of positive ions in a linear mode using an accelerating voltage of 20 kV. 2,5-dihydroxybenzoic acid was used as a matrix.
9
High-Resolution Mass Spectrometry of Oligosaccharides
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High-resolution electrospray ionization mass spectrometry was performed in the negative ion mode using a micrOTOF II instrument (Bruker, Germany). Oligosaccharide samples (~50 ng · L−1) were dissolved in a 1:1 (vol/vol) water-acetonitrile mixture and injected with a syringe at a flow rate of 3 μL · min−1. The capillary entrance voltage was set at 3,200 V, and the interface temperature was 180°C. Nitrogen was used as a drying gas. The mass range was from m/z 50 to m/z 3,500. Internal calibration was done with the ESI Calibrant Solution (Agilent Technologies, USA).
MALDI-TOF mass spectra were registered on a Bruker Ultraflex II mass spectrometer that was equipped with the panoramic delay of ion extraction electronics with the registration of positive ions in a linear mode using an accelerating voltage of 20 kV. 2,5-dihydroxybenzoic acid was used as a matrix.
MALDI-TOF mass spectra were registered on a Bruker Ultraflex II mass spectrometer that was equipped with the panoramic delay of ion extraction electronics with the registration of positive ions in a linear mode using an accelerating voltage of 20 kV. 2,5-dihydroxybenzoic acid was used as a matrix.
10
MALDI-TOF MS Analysis of Tryptic Peptides
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The extracted tryptic peptides obtained from the slice fragments (1 μL) were spotted onto a MALDI target (MTP 600/384 Anchor Chip™; Bruker Daltonics, Germany), mixed with 1 μl of matrix solution (20 mg/ml 2,5-dehydrobenzoic acid in 20% acetonitrile and 0.1% trifluoroacetic acid), and air dried. Three replicates of each sample were placed on the MALDI target. The MS analysis was performed in the reflection/delayed extraction mode at an accelerating voltage of 25 kV with a 135 ns delay, using an Ultraflex II mass spectrometer (Bruker Daltonics). Typically, each mass spectrum was the sum of 100 laser shots. From each target spot, 4–6 mass spectra were acquired. Laser fluency was adjusted to above the desorption threshold of the matrix to obtain the best resolution and the most accurate mass measurements. Signals with a signal/noise ratio > 6 and a maximum of 69 ± 12 peaks per spectrum were used to build peak lists with the SNAP algorithm (FlexAnalysis 2.0, Bruker Daltonics), and the resulting mass spectra were calibrated with trypsin autolysis products (m/z 842.5094 Da and 2211.1046 Da).
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