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.
Maldi tof ms target plate
Manufactured by Bruker
Sourced in Germany, France
The MALDI-TOF MS target plate is a versatile sample holder designed for use with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) instrumentation. The core function of the target plate is to securely hold and position samples for analysis within the mass spectrometer.
Automatically generated - may contain errors
Lab products found in correlation
Microflex lt system, by Bruker (2 mentions)
Maldi biotyper 3, by Bruker (2 mentions)
α cyano 4 hydroxycynnamic acid, by Merck Group (2 mentions)
Trifluoroacetic acid, by Merck Group (2 mentions)
Bacterial test standard, by Bruker (1 mentions)
Flexcontrol software version 3, by Bruker (1 mentions)
α cyano 4 hydroxycinnamic acid matrix solution, by Bruker (1 mentions)
Microflex lt mass spectrometer, by Bruker (1 mentions)
Vitek 2 compact, by bioMérieux (1 mentions)
Microflex spectrometer, by Bruker (1 mentions)
Colombia agar, by bioMérieux (1 mentions)
Microflex lt instrument, by Bruker (1 mentions)
Chocolate agar, by BD (1 mentions)
Macconkey, by BD (1 mentions)
Formic acid, by Merck Group (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
9 protocols using maldi tof ms target plate
1
MALDI-TOF MS Protein Analysis Protocol
2
Glycosyltransferase Activity Assay
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Reactions were prepared containing
0.5 μM KfXYS1, 3 mM UDP-Xyl, 50 mM HEPES sodium
salt pH 7.3, 0.5 mM of oligosaccharide
primer xylobiose (XX), 2′-(4-O-methyl-α-D-glucuronosyl)-xylotriose
(XUX), 3″-arabinofuranosyl-xylotetraose (XAXX), and 3″-(4-O-methyl-α-D-glucuronosyl)-xylotriose (UXX) at various
concentrations (0.5, 0.25, 0.125, 0 mM). Prior to the addition of
UDP-Xyl, 2 μL of the reaction mixture was removed and mixed
with Dowex 50W slurry and incubated for 1 h at room temperature (RT).
Following the addition of UDP-Xyl, reactions were allowed to incubate
at RT for 1 h, at which time 2 μL was removed and mixed with
10 μL of Dowex 50W anion-exchange resin and incubated for 12
h at RT. The samples along with the no UDP-Xyl controls were pelleted,
and 2 μL of the supernatant was mixed with 2 μL of 2,5-dihydroxybenzoic
acid (DHB) (Sigma-Aldrich) dissolved at a concentration of 20 mg/mL
in 50% aqueous methanol on a Bruker MALDI-TOF MS target plate and
allowed to dry. The samples were analyzed with an LT Bruker LT Microflex
spectrometer (Bruker, Billerica, MA), as described previously.11 (link) Positive-ion spectra were recorded with a minimum
of 200 laser shots summed to generate each spectrum.
0.5 μM KfXYS1, 3 mM UDP-Xyl, 50 mM HEPES sodium
salt pH 7.3, 0.5 mM of oligosaccharide
primer xylobiose (XX), 2′-(4-O-methyl-α-D-glucuronosyl)-xylotriose
(XUX), 3″-arabinofuranosyl-xylotetraose (XAXX), and 3″-(4-O-methyl-α-D-glucuronosyl)-xylotriose (UXX) at various
concentrations (0.5, 0.25, 0.125, 0 mM). Prior to the addition of
UDP-Xyl, 2 μL of the reaction mixture was removed and mixed
with Dowex 50W slurry and incubated for 1 h at room temperature (RT).
Following the addition of UDP-Xyl, reactions were allowed to incubate
at RT for 1 h, at which time 2 μL was removed and mixed with
10 μL of Dowex 50W anion-exchange resin and incubated for 12
h at RT. The samples along with the no UDP-Xyl controls were pelleted,
and 2 μL of the supernatant was mixed with 2 μL of 2,5-dihydroxybenzoic
acid (DHB) (Sigma-Aldrich) dissolved at a concentration of 20 mg/mL
in 50% aqueous methanol on a Bruker MALDI-TOF MS target plate and
allowed to dry. The samples were analyzed with an LT Bruker LT Microflex
spectrometer (Bruker, Billerica, MA), as described previously.11 (link) Positive-ion spectra were recorded with a minimum
of 200 laser shots summed to generate each spectrum.
3
Bacterial Identification using Cultivation and Mass Spectrometry
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The organisms were cultivated on a variety of different media (Remel, Lenexa, KS, USA), including trypticase soy agar with 5% sheep blood, chocolate agar, MacConkey agar, mannitol salt agar, and B. cepacia-selective agar. Plates were incubated in ambient air or 5% CO2 at 35°C for 48 hours. After Gram staining, bacteria were further identified using catalase and oxidase tests. A small inoculum from a single colony of each isolate was used to prepare the inocula for biochemical identification by Vitek II Compact® (BioMérieux, Marcy l’Etoile, France), and Phoenix™ (BD Diagnostics, Sparks Glencoe, MD, USA) as recommended by the manufacturer. In parallel, one single colony was directly deposited on a MALDI-TOF MS target plate (Bruker Daltonik GmbH, Bremen, Germany).
4
Standardized Culturomics for Gut Microbiome
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Each stool sample was cultured using the 18 culture conditions of standardized culturomics [8] (link), [9] (link). For each condition, preincubation in the medium of an anaerobic blood culture bottle from BD BACTEC (Becton Dickinson [BD], San Diego, CA, USA) was performed during 30 days with seeding on 5% sheep's blood–enriched Colombia agar (bioMérieux, Marcy l’Étoile, France) every 3 days. Colonies were purified before identification using a Microflex spectrometer and a MTP 96 matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) target plate (Bruker Daltonics, Bremen, Germany) as described previously [14] (link), [15] (link). Each obtained spectrum was matched against the database from Bruker and from the La Timone hospital using MALDI Biotyper 3.0 software by standard pattern matching (with default parameter settings). Matching scores under 1.7 indicated the absence of a match in the database. For these unidentified colonies, the 16S rRNA gene was sequenced using fD1 and rP2 primers as previously described [16] (link). Kim et al. [17] (link) recommended the creation of a new species at the 16S similarity level threshold of 98.65% with the closest species with standing in nomenclature. The corresponding spectra are then incremented in the database and compared to the spectra of closely related species.
5
MALDI-TOF MS Bacterial Identification
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After drying the bacterial pellet on a MALDI‐TOF MS target plate (Bruker Daltonics), 1 μL of alpha‐cyano‐4‐hydroxycinnamic acid (HCCA) matrix solution was placed onto each spot and air‐dried. Triplicate spots were generated for every sample. MALDI‐TOF MS analysis was performed by the Microflex LT system (Bruker Daltonics) with MALDI BIOTYPER 3.3 (Bruker Daltonics) software. Analysis results were presented as an average score of three repeated values for every sample. According to the manufacturer's instructions, a score <1.7 indicates no reliable identification, a score between 1.7 and 1.999 indicates identification to the genus level, and a score ≥2 indicates identification to the species level.
6
MALDI-TOF MS Sample Preparation
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The samples were then centrifuged at 2000×g for 30 s and 1 μl of the supernatant of each homogenate was deposited on a MALDI–TOF MS target plate (Bruker Daltonics, Wissembourg, France) in ten copies. Each deposit was covered with one microlitre of a CHCA matrix suspension, composed of saturated α-cyano-4-hydroxycynnamic acid (Sigma, Lyon. France), 50% acetonitrile (v/v), 2.5% trifluoroacetic acid (v/v) (Aldrich, Dorset, United Kingdom), and high-performance liquid chromatography (HPLC) water to allow for co-crystallisation. After drying for several minutes at room temperature, the target was introduced into the Microflex LT instrument (Bruker Daltonics, Bremen, Germany) for analysis (Fig. 1 c).
7
Rapid Bacterial Identification by MALDI-TOF MS
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Following drying of spotted colonies (routine method) or bacterial pellet (in-house method and SepsiTyper kit) on a MALDI-TOF MS target plate (Bruker Daltonics, Bremen, Germany), 1 μl of alpha-cyano-4- hydroxycinnamic acid (HCCA) matrix solution was placed onto each spot and air-dried. MALDI-TOF MS analysis was performed by Microflex LT system (Bruker Daltonics, Bremen, Germany) with MALDI BIOTYPER 3.3 (Bruker Daltonics) software.
Analysis results are presented as a score. According to manufacturer’s instructions, score < 1.7 indicates no reliable identification, a score between 1.7 and 1.999 indicates identification to the genus level, and a score ≥ 2 indicates identification to the species level.
The methods that were used in the study are summarized in Fig.1 .![]()
Analysis results are presented as a score. According to manufacturer’s instructions, score < 1.7 indicates no reliable identification, a score between 1.7 and 1.999 indicates identification to the genus level, and a score ≥ 2 indicates identification to the species level.
The methods that were used in the study are summarized in Fig.
Flow diagram of the three methods that were used for the identification of positive blood cultures (routine method, in-house method, and SepsiTyper kit)
8
Identification of Microorganisms via MALDI-TOF MS
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Following microorganism growth identification by the BACTEC™ FX system and Gram staining, the blood culture media was subcultured onto solid growth media including blood, MacConkey, and chocolate agar (BD Diagnostics, Sparks, MD) (drop culture). Agar plates were incubated at 36 ± 1 °C in 5% CO2 for 18–24 h. In accordance with clinical data of the patient and/or Gram staining, in case of suspected anaerobic bacteria, the sample was additionally subcultured on CDC and blood-amikacin agars (Hy-Laboratories Ltd., Israel), which were incubated at 37 °C for 5 days, under anaerobic conditions. Whenever Gram stain indicated presence of yeasts, the sample was additionally subcultured on CHROMagar Candida (Hy-Laboratories Ltd., Israel), which was incubated at 37 °C for 48 h. Following incubation, isolated colonies were spotted onto a MALDI-TOF MS target plate (Bruker Daltonics, Bremen, Germany) and subjected to MALDI-TOF MS analysis, as described below.
9
Mosquito Protein Extraction for MALDI-TOF MS
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For MALDI-TOF MS analysis, the legs of each mosquito were placed in a 1.5 mL Eppendorf tube and ground with Tissue Lyser (Qiagen, Hilden, Germany) in a mix solution containing 15 µL of 70% (v/v) formic acid (Sigma-Aldrich, St. Louis, MO, USA) and 15µl of 50% (v/v) acetonitrile (Fluka, Buchs, Switzerland) and a pinch of glass beads (Sigma, Lyon, France) for three minutes divided into three 60 s cycles at a frequency of 30 Hz [28 (link)]. The samples were then centrifuged for one minute at 10,000 rpm and 1 µL of the supernatant from each homogenate was deposited in four replicates on a MALDI-TOF MS target plate (Bruker Daltonics, Wissembourg, France). Each spot was then coated with 1 µL of a matrix solution of α-cyano-4-hydroxycynnamic acid (CHCA) composed of 50% acetonitrile (v/v), 2.5% trifluoroacetic acid (v/v) (Aldrich, Dorset, UK), saturated α-cyano-4-hydroxycynnamic acid (Sigma, Lyon, France) and high-performance liquid chromatography (HPLC)-grade water [29 (link)]. After drying for a few minutes at room temperature, the target plate was introduced into the MALDI-TOF Microflex LT apparatus (Bruker Daltonics, Bremen, Germany) for analysis. Anopheles coluzzii legs reared in our laboratory were used as a positive control.
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