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.
Flexcontrol software version 3
Manufactured by Bruker
Sourced in Germany
FlexControl software version 3.3 is a core software package developed by Bruker for controlling various analytical instruments. The software serves as the central interface for instrument operation, data acquisition, and data processing. It provides a comprehensive set of tools to configure, run, and monitor experiments across Bruker's product line.
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Lab products found in correlation
Microflex lt mass spectrometer, 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)
Flexanalysis software version 3, by Bruker (1 mentions)
Ultraflex 3, by Bruker (1 mentions)
α cyano 4 hydroxycinnamic acid matrix solution, by Bruker (1 mentions)
Maldi tof ms target plate, by Bruker (1 mentions)
Maldi biotyper real time classification version 3, by Bruker (1 mentions)
Maldi tof ms, by Bruker (1 mentions)
Surfactin, by Merck Group (1 mentions)
Iturin a, by Merck Group (1 mentions)
Ultraflex 3 maldi tof, by Bruker (1 mentions)
α cyano 4 hydroxycinnamic acid, by Bruker (1 mentions)
Peptide calibration standard 2, by Bruker (1 mentions)
Maldi tof ms biotyper system, by Bruker (1 mentions)
7 protocols using flexcontrol software version 3
1
Mass Spectrometry Analysis of Peptide Purity
2
MALDI-TOF MS Identification of Pythium insidiosum
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A 0.5-mL volume of the extracted protein-containing supernatant (see above) was spotted onto a clean ground steel target plate (Bruker Daltonics, Germany) in 40 replicates (for generating a MALDI-TOF MS database of P. insidiosum) or 5 replicates (for assessing the MALDI-TOF MS for identification of P. insidiosum), air dried at room temperature, and then overlaid with 0.5 mL of the matrix solution (5 mg/mL α-cyano-4-hydroxy-cinnamic acid in 70% acetonitrile and 0.1% trifluoroacetic acid). After the matrix solution was air dried at room temperature, the sample was promptly analyzed, using an ultrafleXtreme mass spectrometer and the FlexControl software version 3.0 (Bruker Daltonics, Germany), with the following
3
MALDI-TOF MS Bacterial Identification
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One microliter of Bacterial Test Standard (BTS; Bruker) was applied to a clean spot on the same MALDI stainless steel target and allowed to air dry. After drying, the BTS spot was overlaid with 1 μL of HCCA matrix solution within 30 min and allowed to air dry at room temperature.
The target was loaded into the microflex® LT mass spectrometer (Bruker) for spectral acquisition. A cumulative spectrum is generated using six sets of 40 laser shots for each spot (for a cumulative total of 240 shots per spot) with a nitrogen laser frequency of 60-Hz, a voltage of 20 kv, and mass to charge ratio of ions range of 2–20 k Dalton. The instrument was calibrated with the BTS using the AutoXecute algorithm in flexControl® software version 3.4 (Bruker) to manufacturer specifications. For calibration, the automatic laser position was manually overridden by moving the laser to the left side of the spot. The instrument was considered to pass calibration with eight reference mass peaks successfully assigned within ± 300 parts per million.
The target was loaded into the microflex® LT mass spectrometer (Bruker) for spectral acquisition. A cumulative spectrum is generated using six sets of 40 laser shots for each spot (for a cumulative total of 240 shots per spot) with a nitrogen laser frequency of 60-Hz, a voltage of 20 kv, and mass to charge ratio of ions range of 2–20 k Dalton. The instrument was calibrated with the BTS using the AutoXecute algorithm in flexControl® software version 3.4 (Bruker) to manufacturer specifications. For calibration, the automatic laser position was manually overridden by moving the laser to the left side of the spot. The instrument was considered to pass calibration with eight reference mass peaks successfully assigned within ± 300 parts per million.
4
MALDI-TOF MS Protein Analysis Protocol
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The protein extracts (see the “Protein extraction ” section) mixed with formic acid and acetonitrile were centrifuged at 18,312 × g for 2 min. One microliter of the clear supernatant was spotted onto the MALDI-TOF MS target plate (Bruker Daltonics, Bremen, Germany) then allowed to dry completely before covering it with 1 µL of α-cyano-4-hydroxycinnamic acid matrix solution (Bruker Daltonics) composed of saturated α-cyano-4-hydroxycinnamic acid, 50% (v/v) acetonitrile, 2.5% (v/v) trifluoroacetic acid and 47.5% (v/v) LC–MS grade water. The protein extracts of each sample were spotted onto the MALDI-TOF MS target plate at eight different spots, and each spot was measured four times to assure reproducibility. Hence, a total of 32 raw spectra per sample were generated using FlexControl® software version 3.4 (Bruker Daltonics). The bacterial test standard (Bruker Daltonics), which is an extract of Escherichia coli spiked with two high molecular weight proteins, was used to calibrate the mass spectrometer. After drying at room temperature, the MALDI target plate was placed into a Microflex LT Mass Spectrometer (Bruker Daltonics) for the measurements.
5
Identification of Nepenthes Pitcher Bacteria
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Bacteria were isolated from highland Nepenthes pitcher fluid (sample H1) and maintained using Luria-Bertani (LB) medium at 28 °C. The identity of the culturable bacteria was identified using MALDI-TOF MS (Bruker, Germany) equipped with Bruker FlexControl software version 3.3 and Bruker MALDI Biotyper Real Time Classification (RTC) version 3.1. This bacterial identification was performed according to the direct transfer procedure from Bruker45 (link). Protein from the bacterial cell was measured by MALDI-TOF MS and the spectra generated were compared to the reference database for bacterial identification. Results with the log (score) value equal or higher than 2 indicate high confidence identification and is tabulated in Table 1 .
The bacterial identities were confirmed molecularly by phylogenetic analysis on their 16S rDNA genes sequences. Bacterial genomic DNA were extracted using QIAamp DNA mini kit according to manufacturer instruction. 16S rDNA genes were amplified with 27F and 1525R primer pair46 (link). Molecular identities of the isolates were identified based on the phylogenetic analysis of the 16S rDNA gene sequences using MEGA (v6.06)47 (link).
The bacterial identities were confirmed molecularly by phylogenetic analysis on their 16S rDNA genes sequences. Bacterial genomic DNA were extracted using QIAamp DNA mini kit according to manufacturer instruction. 16S rDNA genes were amplified with 27F and 1525R primer pair46 (link). Molecular identities of the isolates were identified based on the phylogenetic analysis of the 16S rDNA gene sequences using MEGA (v6.06)47 (link).
6
MALDI-TOF-MS Characterization of Biosurfactants
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The chromatographic fraction containing the bioactive biosurfactants produced by TR47II (F4) was solubilized in ethanol (2.5 mg mL−1) and characterized by MALDI-TOF-MS. The matrix used was α-cyano-4-hydroxycinnamic acid (Bruker Daltonics), solubilized in acetonitrile 50% (v/v), and acidified with trifluoroacetic acid 0.1% (v/v), to a final concentration of 10 mg mL−1. Then, 1 μl of the sample and 1 μl of the matrix were applied and homogenized at each spot of the steel plate. For calibration of the MS analysis method, standard peptides (Peptide Calibration Standard II) (Bruker Daltonics) were used. The MS spectra were acquired in an MALDI-TOF Ultraflex III (Bruker Daltonics) spectrometer employing the reflective and positive mode, with an average detection range of 500–3400 Da. All data were managed by Flexcontrol software, version 3.3 (Bruker Daltonics), and the spectra resulting from the MS were processed using the FlexAnalysis application, version 3.3 (Bruker Daltonics). Commercial standards of surfactin, iturin A, and fengycin A lipopeptides (Sigma-Aldrich) were used in a concentration of 1 mg mL−1 in ethanol. The spectra were analyzed based on the similarity with the spectra of commercial lipopeptide standards and based on literature (Romero et al. 2007 (link); Pathak and Keharia 2014 (link); Yang et al. 2015 (link)).
7
Bacterial Identification from Throat Swabs
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Throat swabs for bacterial identification were inoculated on Tryptic Soy Agar with 5% sheep’s blood (TSAB) (Isolab Sdn Bhd, Malaysia) and incubated for 24 hrs at 37°C with 5% CO2. One to five single colonies with distinct morphologies (shape, colour, size and haemolytic pattern) were sub-cultured on TSAB agar and screened on the Microflex matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) Biotyper system (Bruker Daltonik GmbH, Germany) using the Bruker FlexControl software version 3.3 (Build 108) and analysed using the Bruker MALDI Biotyper Real Time Classification software (Version 3.1, Build 65).
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