The MS spectra were then exported to flex Analysis v3.3, ClinProTools v2.2 and MALDI-Biotyper v3.0. (Bruker Daltonics) software for data processing (smoothing, baseline subtraction, peak picking). The quality of MS spectra was evaluated by visualization of spectra obtained from the four spots for each sample with the flex Analysis v3.3 software (Bruker Daltonics). Cluster analyses (MSP dendrogram) and principal component analysis (PCA) were performed to verify intra-species reproducibility and inter-species specificity as well as variability within different castes (soldiers and workers) of the same species. Cluster analyses were performed based on the comparison of the MSPs given by the MALDI-Biotyper v3.0. software and grouped according to the mass profile of the proteins (i.e., their mass signals and intensities) and it reflects how tick specimens are related to each other. The setting parameters were as follows: distance measure by correlation, linkage by average; the score threshold value for a single organism was 300 (arbitrary unit) and for related organisms was 0 (arbitrary unit).
Flexanalysis v 3
Manufactured by Bruker
Sourced in Germany, United States
FlexAnalysis v.3.4 is a software application developed by Bruker for the analysis of data from various analytical instruments. It provides a comprehensive suite of tools for data processing, visualization, and interpretation. The software is designed to be user-friendly and versatile, catering to the needs of researchers and scientists across various fields.
Automatically generated - may contain errors
Lab products found in correlation
Flexcontrol v3, by Bruker (7 mentions)
Clinprotools v3, by Bruker (7 mentions)
Clinprotools v 2, by Bruker (6 mentions)
Fleximaging software v4, by Bruker (5 mentions)
Maldi biotyper v 3, by Bruker (5 mentions)
Flexcontrol software, by Bruker (4 mentions)
Ultraflex 2, by Bruker (4 mentions)
Ultraflextreme, by Bruker (3 mentions)
Flexcontrol 3, by Bruker (3 mentions)
Microflex lt maldi tof mass spectrometer, by Bruker (3 mentions)
Clinprotools 2, by Bruker (3 mentions)
Polished steel target plate, by Bruker (2 mentions)
Microflex lrf, by Bruker (2 mentions)
Prism 7, by GraphPad (2 mentions)
Maldi tof microflex lt, by Bruker (2 mentions)
Fleximaging v4, by Bruker (2 mentions)
Maldi biotyper sirius system, by Bruker (2 mentions)
Bacterial test standard, by Bruker (2 mentions)
Anchorchip target plate, by Bruker (2 mentions)
Microflex lt maldi tof ms, by Bruker (1 mentions)
54 protocols using flexanalysis v 3
1
MALDI-TOF MS Profiling of Tick Species
2
MALDI-TOF Analysis of Plasmodium Samples
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Samples were analysed using a Microflex LT MALDI-TOF (Bruker Daltonics) and FlexContol v.3.4 (Bruker Daltonics). The following settings were applied: Sampling range 1000–20,000 Da, laser power 30–50% of maximum, accumulation of 800 shots pr. spectra, movement pattern random walk, maximum 20 shots allowed pr. raster position, automated peak quality evaluation from 4000 to 10,000 Da, standard exclusion of the one largest peak in the mass range, minimum peak resolution 200 a.u., fuzzy control peptide mode with high signal intensity, and automated termination if 100 consecutive shots failed. Resulting spectra were visually evaluated in FlexAnalysis v.3.4 (Bruker Daltonics). Baseline subtraction and smoothing was applied once before visual inspection. Peaks found in Plasmodium samples but not in the negative controls were noted. As a quality control, spectra were further compared to the existing bacterial database with the standard built-in automated comparison tool. MALDI-TOF calibration and detector check was preformed according to the manufacturer’s instructions before initiating the study.
3
MALDI-TOF-MS Sample Preparation with CHCA and DHB
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A saturated solution of α-cyano-4-hydroxycinnamic acid (CHCA, 20 mg) in acetone (500 μL) was prepared as Mix 1. A precursor saturated solution of CHCA (20 mg) in 70% acetonitrile with 5% formic acid (500 μL) was prepared alongside a saturated solution of 2,5-dihydroxybenzoic acid (DHB, 20 mg, Sigma-Aldrich) in 70% acetonitrile with 0.1% trifluoroacetic acid (500 μL). Solutions were prepared at room temperature and vortexed thoroughly for 60 seconds before use. DHB (100 μL) and CHCA (100 μL) solutions were then combined to prepare Mix 2. Mix 1 (0.5 μL) was spotted onto a polished steel target plate (Bruker) and evaporated quickly to leave a thin layer of CHCA. A 0.5 μL aliquot of protein sample (typically 10 μM in PBS) was spotted directly onto the layer. Then, 0.5 μL of Mix 2 was added to the liquid droplet and allowed to dry. Where required, antibody samples were reduced by incubation with dithiothreitol (DTT, 10 mM) at 60 °C for 30 min.
A Bruker Microflex LRF was used to acquire the MALDI-TOF-MS data, in linear positive mode (laser 60 Hz, ion source 1: 19.5 kV, ion source 2: 18.15 kV, lens: 7.00 kV, pulsed ion extraction 240 ns, detector gain 2850 V). Data were processed using Bruker flexAnalysis v3.4.
A Bruker Microflex LRF was used to acquire the MALDI-TOF-MS data, in linear positive mode (laser 60 Hz, ion source 1: 19.5 kV, ion source 2: 18.15 kV, lens: 7.00 kV, pulsed ion extraction 240 ns, detector gain 2850 V). Data were processed using Bruker flexAnalysis v3.4.
4
Enzymatic Hydrolysis and Analysis of α-1,4-GalNAc Oligosaccharides
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α-1,4-GalNAc oligosaccharides were obtained by a Sph3 partial hydrolysis. A. fumigatus biofilms were incubated with 5 nM Sph3 for 1 h at room temperature, solubilized oligosaccharides were then further purified on a Sep-pak C18 cartridge. In brief, cartridges were conditioned using absolute ethanol followed by water. Samples were then loaded onto the cartridge before washing and eluting using a 1% (v/v) step gradient of ACN from 1 to 4% (v/v). 3% (v/v) ACN fraction was used for the specificity study.
Purified oligosaccharide fractions were mixed with Agd3 WT or its mutants as indicated, and the samples analyzed by MS at multiple time points. Products of enzymatic reaction were diluted in 0.2% (v/v) TFA before being spotted on the MALDI-TOF plate in a ratio 1:1 (v:v) with 5 mg/mL DHB matrix reconstituted in ACN: 0.2% (v/v) TFA (70:30, v–v). Spectra were recorded on a Bruker UltrafleXtreme in positive reflector mode and an accumulation of 5000 laser shots. MALDI-TOF MS/MS experiments were performed using the same mass spectrometer. Data was collected and analyzed using Bruker Flex Control v.3.4 and Bruker Flex Analysis v.3.4.
Purified oligosaccharide fractions were mixed with Agd3 WT or its mutants as indicated, and the samples analyzed by MS at multiple time points. Products of enzymatic reaction were diluted in 0.2% (v/v) TFA before being spotted on the MALDI-TOF plate in a ratio 1:1 (v:v) with 5 mg/mL DHB matrix reconstituted in ACN: 0.2% (v/v) TFA (70:30, v–v). Spectra were recorded on a Bruker UltrafleXtreme in positive reflector mode and an accumulation of 5000 laser shots. MALDI-TOF MS/MS experiments were performed using the same mass spectrometer. Data was collected and analyzed using Bruker Flex Control v.3.4 and Bruker Flex Analysis v.3.4.
5
MALDI Data Analysis Pipeline for Lipid Profiling
6
MALDI-TOF and Orbitrap MS Protein Identification
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Data processing of the spectra from MALDI-TOF was performed with flexAnalysis v.3.4 (Bruker Daltonik GmbH). The generated peptide mass list (mass + H+) of the major peaks from MALDI-TOF analysis was submitted to a database search (NCBI or SWISS-PROT) using MS-fit search engines. Parameters were set as species (human), mass tolerance (50 ppm), maximum missed cleavages by trypsin ≤1, fixed modification (carbamidomethylation), dynamic modification (oxidation of methionine), N-terminal glutamine to pyroglutamate, and N-terminus acetylation.
Data from the Orbitrap mass spectrometer were searched with MaxQuant version 1.5 with trypsin as a digestion enzyme against a human taxonomy of the SwissProt database (release February 2016). The following parameters were used: maximum number of missed cleavages 2; fragment ion mass tolerance 0.5 Da; parent ion mass tolerance 6 ppm; fixed modification – carbamidomethylation of cysteine; and the variable modifications–N-terminal acetylation and methionine oxidation. Data were filtered at 1% false discovery rate. Identifications were based on a minimum of two unique peptides.
Data from the Orbitrap mass spectrometer were searched with MaxQuant version 1.5 with trypsin as a digestion enzyme against a human taxonomy of the SwissProt database (release February 2016). The following parameters were used: maximum number of missed cleavages 2; fragment ion mass tolerance 0.5 Da; parent ion mass tolerance 6 ppm; fixed modification – carbamidomethylation of cysteine; and the variable modifications–N-terminal acetylation and methionine oxidation. Data were filtered at 1% false discovery rate. Identifications were based on a minimum of two unique peptides.
7
MALDI Lipid Profiling of Ischemia-Reperfusion
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Matrix-assisted laser desorption/ionization data were processed using FlexAnalysis v3.4 (Bruker Daltonics, Billerica, MA, USA) and visualized with FlexImaging software v4.0 (Bruker Daltonics, Billerica, MA, USA). Results were analyzed using ClinProTools v3.0 (Bruker Daltonics, Billerica, MA, USA). Lipids were identified by their m/z values using the literature and LIPID MAPS (http://www.lipidmaps.org/tools/index.html ) and confirmed by tandem mass spectrometry (MS/MS) in the LIFT-TOF/TOF mode22 (link). Intergroup comparisons were displayed using data bar plots (Microsoft Excel 2016 Conditional Formatting; Microsoft Corporation, Redmond, WA, USA) and a heatmap. For the data bar plots, intergroup differences were analyzed using T-tests with a 5% false discovery rate (GraphPad Prism 7, La Jolla, CA, USA). For the heatmap, lipid profiles based on average intensities of expressed lipid ions and those associated with different durations of ischemia–reperfusion were compared in GeneCluster 3.023 (link). Hierarchical clustering was applied, and the dendrogram was displayed using Java TreeView23 (link)-25 (link, link) . Intergroup differences were compared by one-way analysis of variance (ANOVA) and the Tukey post hoc repeated measures test. χ2 tests assessed proportional alterations in lipid subtypes after different durations of I/R (GraphPad Prism 7, La Jolla, CA, USA).
8
Protein Mass Profiling via MALDI-TOF MS
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Protein mass profiles were acquired using FlexControl 3.4 software (Bruker Daltonics, Germany). The spectra were recorded in the linear positive-ion mode (ion source 1 = 19.5 kV, ion source 2 = 18.2 kV, detector voltage = 2872 V, pulsed ion extraction = 200 ns) with a mass range from 2 to 20 kDa27 (link),60 (link). Each spectrum corresponded to ion accumulation of 10,000 laser shots randomly distributed on the spot. The spectra obtained were processed with default parameters [Savitsky-Golay smoothing (5 Da, two runs), top hat algorithm for baseline subtractions, peak picking (150 maximum, S/N >= 3), peak width = 5 m/z] using FlexAnalysis v.3.4 software (Bruker Daltonics, Germany).
9
Vibrio Species Database Construction
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Baseline subtraction and smoothing were performed after importing the spectra into flexAnalysis v. 3.4 (Bruker). Next, we searched the spectra set for flatline spectra, sweet spot outliers and anomalies, removing such spectra by closing out of the set. A minimum of 18 spectra were required for main spectra projections (MSP) creation; where necessary the measurement was repeated using a new sample preparation to achieve this. In the peak shift of individual masses, we selected peaks from 3,000 to 10,000 Da in steps of 1,000. The allowed peak shift between the spectra with the smallest and the largest mass was 500 ppm. Finally, we selected the remaining spectra in flexAnalysis v. 3.4 and used these data to create the MSP through MALDI BioTyper Compass Explorer v. 4.1 software. All MSPs were registered in our in-house database, PVBase. In addition, we established separate databases of different sizes for V. parahaemolyticus, V. cholerae, V. alginolyticus, V. harveyi, and V. vulnificus to observe the changes in the highest matching scores identified by different sizes of MSP present.
10
MALDI-TOF MS Mycobacteria Identification
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We adapted the Microflex LT MALDI-TOF MS (Bruker Daltonics) to acquire the spectra in the linear positive-ion mode (with a laser frequency of 60 Hz and a mass/charge ratio [m/z] of ∼2 to 2,000 Da). The protein profile was respectively obtained and analyzed using FlexControl v3.4 and FlexAnalysis v3.4 software (both from Bruker Daltonics). Mycobacteria Library v2.0 was used; it contains 313 mycobacterial protein profiles, representing 131 species.
The quality of spectra generated by the mycobacterial extraction protocol for MALDI-TOF MS was evaluated according to the following criteria. The software assigned a score of ∼0 to 3 and classified the results into three categories: reliable (species level; ≥2), probable (genus level; ∼1.7 to 1.999), and nonidentifiable (<1.7). Identification was proven when a score of ≥1.70 was obtained and when the identification matched at least 5 of the top 10 species identifications provided by MALDI-TOF MS. We considered a range of ∼2.0 to 3.0 as acceptable, and scores of ∼1.7 to 2.0 were considered consistent when the same identification was repeated in most of the 10 possibilities provided by the project. Lower scores (<1.7) were reported as unreliable identification (35 (link)– (link)37 (link)).
The quality of spectra generated by the mycobacterial extraction protocol for MALDI-TOF MS was evaluated according to the following criteria. The software assigned a score of ∼0 to 3 and classified the results into three categories: reliable (species level; ≥2), probable (genus level; ∼1.7 to 1.999), and nonidentifiable (<1.7). Identification was proven when a score of ≥1.70 was obtained and when the identification matched at least 5 of the top 10 species identifications provided by MALDI-TOF MS. We considered a range of ∼2.0 to 3.0 as acceptable, and scores of ∼1.7 to 2.0 were considered consistent when the same identification was repeated in most of the 10 possibilities provided by the project. Lower scores (<1.7) were reported as unreliable identification (35 (link)– (link)37 (link)).
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