Once the purity of each peptide had been determined, the set of seven‐point calibration standards was then analysed using two different mass spectrometers and two ionisation sources. The two instruments used were the Bruker ultraflex III (TOF) and the Bruker solariX XR 9.4 T (FT‐ICR). The ultraflex was used with its fixed MALDI source in positive ion mode with 800 laser shots per sample acquisition. The data were acquired using flexcontrol software version 3.0 (Bruker Daltonics). Each spot was analysed in reflector mode using a smartbeam™ Nd:YAG laser (355 nm). Spectra were analysed using flexAnalysis software version 3.0 (Bruker Daltonics). The solariX was operated with either an ESI (direct infusion) or MALDI source in positive ion mode (800 laser shots per sample acquisition) with a smartbeam™ Nd:YAG laser (355 nm). Spectra were acquired using the solariXcontrol software and processed with DataAnalysis version 4.2 (Bruker Daltonics). Each sample was analysed in triplicate and the average values of the peak intensities are reported.
Flexanalysis software version 3
Manufactured by Bruker
Sourced in Germany
FlexAnalysis software version 3.4 is a data analysis tool developed by Bruker. It provides functionality for processing and analyzing data from various Bruker analytical instruments.
Automatically generated - may contain errors
Lab products found in correlation
Maldi biotyper software version 3, by Bruker (3 mentions)
Flexcontrol 3, by Bruker (2 mentions)
Bacterial test standard, by Bruker (2 mentions)
Dataanalysis version 4, by Bruker (1 mentions)
Solarix xr 9.4t, by Bruker (1 mentions)
Flexcontrol software version 3, by Bruker (1 mentions)
Ultraflex 3, by Bruker (1 mentions)
Ppsq 31b, by Shimadzu (1 mentions)
Ultraflextreme tof tof mass spectrometer, by Bruker (1 mentions)
Peptide calibration mix 2, by Bruker (1 mentions)
Ultraflex 3 maldi tof tof mass spectrometer, by Bruker (1 mentions)
Ag 50w x8 resin, by Bio-Rad (1 mentions)
2 5 dihydroxybenzoic acid, by Bruker (1 mentions)
Autoflex speed, by Bruker (1 mentions)
Microflex instrument, by Bruker (1 mentions)
Biotyper system, by Bruker (1 mentions)
Microflex, by Bruker (1 mentions)
Vitek ms, by Bruker (1 mentions)
Flexanalysis software, by Bruker (1 mentions)
Maldi biotyper 3, by Bruker (1 mentions)
19 protocols using flexanalysis software version 3
1
Mass Spectrometry Analysis of Peptide Purity
2
MALDI-TOF Mass Spectrometry Analysis
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The mass spectra were evaluated with FlexAnalysis software version 3.0 (Bruker Daltonics® Bremen, Germany). The automated data analysis was processed with MALDI Biotyper software version 3.0 (Bruker Daltonics® Bremen, Germany). The list of the best peaks of the spectrum was created automatically by the software after smoothing, normalization and baseline subtraction.
3
MALDI-TOF/TOF Peptide Fragmentation and Sequencing
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MALDI in-source decay was performed using an Ultraflextreme TOF/TOF mass spectrometer controlled by the FlexControl 3.3 software (BrukerDaltonics). Spectra were acquired in positive reflectron ion mode with 2000 laser shots accumulated, and the laser power was set with an increase in 20% of fluensce to fragment protein in the source of the mass spectrometer using a 20 mg/mL solution of 1,8-diaminonaphtalen (DAN) matrix enabling the generation of hydrogen radicals that break the peptide backbone producing c-ions and z-ions from 1000 to 5000 Da. The generated fragment ions allowed the sequence annotation of the peptides. The mass spectrometer parameters were set according to the manufacturer’s settings for optimal acquisition performance. Sequences were analyzed on Flex Analysis software, version 3.0 (BrukerDaltonics). Mass spectrometer calibration was performed using the c-ions of 1 pmole of BSA spotted with the DAN matrix.
N-terminal sequencing of the peptide present on the active fraction was performed by Edman degradation. Approximately 600 pmol of the sample was loaded on a glass fiber membrane pretreated with 10 µL of biobrene and then analyzed using an automatic sequencer model PPSQ 31B (Shimadzu, Kyoto, Japan). The sequence was compared with the Uniprot database and with the sequence obtained by MS.
N-terminal sequencing of the peptide present on the active fraction was performed by Edman degradation. Approximately 600 pmol of the sample was loaded on a glass fiber membrane pretreated with 10 µL of biobrene and then analyzed using an automatic sequencer model PPSQ 31B (Shimadzu, Kyoto, Japan). The sequence was compared with the Uniprot database and with the sequence obtained by MS.
4
MALDI-TOF-MS Identification of Bacterial Cells
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The extraction procedure using ethanol and formic acid was used to prepare the samples for MALDI-TOF-MS identification. Two full loops of bacterial cells containing 1 μl were resuspended in an test tube with 300 μl of distilled water. 900 μl of absolute ethanol was mixed into the test tube and the mixture was centrifuged (15 000 rpm/2 min). The supernatant was pipetted off 50 µl of 70% formic acid and 50 µl of acetonitrile were added to the pellet before analysis and the mixed mixture was centrifuged again (15 000 rpm/2 min). Subsequently, 1.0 μl of supernatant was applied to a MALDI plate, which, after drying, was overlaid with 1.0 μl of a saturated solution of α-cyano-4hydroxycinnamic acid (HCCA) in 50% acetonitrile and 2.5% trifluoroacetic acid (Bruker Daltonics, 2008) . The analysis of the results was performed in an Ultraflex III device. The obtained results were processed using Flex Analysis software, version 3.0 and evaluated using BioTyper software, version 1.1 (BRUKER DALTONICS, Massachusetts, USA).
5
MALDI-TOF MS-based Identification of Pythium insidiosum
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Based on the pattern matching approach, the FlexAnalysis software version 3.0 (Bruker Daltonics, Germany) was used to view and process spectra, including spectral smoothness and baseline subtraction, of each sample. Twenty-four best mass spectra were selected (based on peak positions and intensities) from a set of 40 generated spectra of each database strain. These spectra were used to construct a main spectral profile (MSP), using the MALDI Biotyper OC software version 3.1 (Bruker Daltonics, Germany) with the default setting. MSPs of all P. insidiosum database strains were then added to the reference Bruker MALDI Biotyper database DB4613 (Bruker Daltonics, Germany), which includes 4,613 MSPs from bacteria (n = 4,274), fungi (n = 331), archaea (n = 7), and green algae (n = 1).
Five mass spectra, newly generated from each strain of P. insidiosum (Table 1), were used to assess the MALDI-TOF MS-based identification. Depending on mass spectrum similarity, an identification score (range, 0.00-3.00) was calculated by the MALDI Biotyper software version 3.0 (Bruker Daltonics, Germany), where a score of < 1.70 suggests an unreliable identification of the microorganism; a score of ! 1.70 represents a reliable identification at genus level; and a score of ! 2.00 indicates a reliable species-level identification.
Five mass spectra, newly generated from each strain of P. insidiosum (Table 1), were used to assess the MALDI-TOF MS-based identification. Depending on mass spectrum similarity, an identification score (range, 0.00-3.00) was calculated by the MALDI Biotyper software version 3.0 (Bruker Daltonics, Germany), where a score of < 1.70 suggests an unreliable identification of the microorganism; a score of ! 1.70 represents a reliable identification at genus level; and a score of ! 2.00 indicates a reliable species-level identification.
6
MALDI-TOF Mass Spectrometry Protocol
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Mass spectra were obtained with an Ultraflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonik, Bremen). The instrument was operated in positive ionization and reflectron mode. The mass range was set to 600–5000 Da. For each sample, 1000 spectra were accumulated using a laser power of 30% and a laser frequency of 100 Hz. Delayed extraction (490 µs delay) was applied. For external mass calibration, Peptide Calibration Mix II (Bruker Daltonik, Bremen) was employed. The flexAnalysis software version 3.3 (Bruker Daltonik, Bremen) was used to determine peak areas.
7
MALDI-TOF MS Analysis of Lithium Adducts
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For matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), an Ultraflex workstation using FlexControl 3.3 (Bruker Daltonics) equipped with a nitrogen laser of 337 nm was used. The pulsed ion extraction was set on 80 ns. Ions were accelerated to a kinetic energy of 25 kV and detected in positive reflector mode with a set reflector voltage of 26 kV. The lowest laser energy required was used to obtain a good signal-to-noise ratio. A total of 200 spectra were collected for each measurement. The mass spectrometer was calibrated using a mixture of maltodextrins (Avebe, Veendam, The Netherlands) in a mass range (m/z) of 500–2,500. The peak spectra were processed by using FlexAnalysis software version 3.3 (Bruker Daltonics). Prior to analysis, samples were desalted by adding AG 50 W-X8 Resin (Bio-Rad Laboratories). To obtain lithium (Li) adducts, the supernatant was dried under nitrogen and re-suspended in 20 mM LiCl [28 (link)]. Each lithium-enriched sample of a volume of 1 µL was mixed with 1 µL of matrix solution (12 mg mL−1 2,5-dihydroxy-benzoic acid (Bruker Daltonics) in 30% (v/v) acetonitrile in H2O), applied on an MTP 384 massive target plate (Bruker Daltonics) and dried under a stream of warm air.
8
MALDI-ToF–MS Analysis of Diluted Samples
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Prepared samples were diluted 20-fold with deionized water, then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF–MS). A Bruker Autoflex Speed mass spectrometer, it was used for analysing the samples using 2,5-dihydroxybenzoic acid as matrix mass spectra, using Bruker Flex analysis software version 3.3 and were annotated manually.
9
Phage Protein Identification by Mass Spectrometry
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Phage particles purified by CsCl-gradient centrifugation were mixed with a gel electrophoresis sample buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 200 mM dithiothreitol), boiled for 10 min, and subjected to SDS-polyacrylamide gel electrophoresis (8–16% gradient). Protein bands were visualized using InstantBlue (Expedeon Protein Solutions Ltd., Cambridge, UK). To determine the identity of the protein in a gel, protein bands were excised and subjected to peptide mass fingerprinting using a Microflex instrument (Bruker Corporation, Billerica, MA, USA) for de novo peptide analysis by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The spectra were recorded in the linear mode in a detection range of 500–3500 m/z and subsequently analyzed using the flexAnalysis software version 3.4 (Bruker Corporation). The data were searched using the MASCOT peptide mass fingerprinting search program, against a local database of possible peptide spectra deduced from the Ahp2 genome sequence.
10
MALDI-TOF MS Profiling of Schistosoma Worms
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Raw spectra were visualized using FlexAnalysis software version 3.4 (Bruker Daltonics). The spectra were edited, i.e., all flatlines and outlier peaks were removed, intensities were smoothed, and peak shifts within replicated spectra were set at 300 p.p.m. After this editing step, spectra of four mixed (males/females) adult worm samples (two S. mansoni, two S. japonicum) from both of the storage media (RNAlater, ethanol), comprising at least 27 remaining spectra each, were randomly selected for the creation of reference spectra (main spectra profiles; MSPs). These MSPs were created using the automatic function of MALDI Biotyper Compass Explorer® software version 3 (Bruker Daltonics). The newly created MSPs of both Schistosoma species were included in a previously developed in-house MALDI-TOF MS database for helminth identification, which already contained MSPs from different helminths such as cestodes (e.g., Taenia saginata) and trematodes (e.g., Fasciola spp.) [10 (link), 17 ].
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