All amino acids and resins were purchased from Novabiochem (Merck Millipore) and Carbosynth. All other synthetic reagents were obtained from Novabiochem, Sigma-Aldrich, or Carl Roth. Peptide synthesis was performed on a MultiPep RSi peptide synthesizer (Intavis Bioanalytical Instruments AG). Peptides were purified using a 1260 Infinity II preparative HPLC system (Agilent Technologies) with a VP 250/10 NUCLEODUR 100-5 C18 ec column (Macherey-Nagel) running a general gradient of 10–70% acetonitrile (ACN) in H2O and validated using a 1260 Infinity I HPLC System coupled to a 6120 Quadrupole LC-MS (Agilent Technologies) with an EC 250/4 NUCLEODUR 100-5 C18 ec column (Macherey-Nagel) with a general gradient of 10–90% ACN in H2O, and additionally a Microflex LT MALDI (Bruker) was used for the analysis.
Maldi microflex lt
Manufactured by Bruker
Sourced in Germany
The MALDI Microflex LT is a matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometer designed for basic analytical applications. It provides reliable and reproducible mass spectra of small molecules, peptides, and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Biotyper database, by Bruker (4 mentions)
6120 quadrupole lc ms, by Agilent Technologies (2 mentions)
Vp 250 10 nucleodur 100 5 c18 ec column, by Macherey-Nagel (2 mentions)
1260 infinity 1 hplc system, by Agilent Technologies (2 mentions)
Ec 250 4 nucleodur 100 5 c18 ec column, by Macherey-Nagel (2 mentions)
Multipep rsi peptide synthesizer, by Intavis (2 mentions)
Wasp dt walk away specimen processor, by Copan (2 mentions)
Brilliance uti agar, by Thermo Fisher Scientific (2 mentions)
Cm0115, by Thermo Fisher Scientific (1 mentions)
Maldi biotyper database, by Bruker (1 mentions)
Maldi biotyper software, by Bruker (1 mentions)
Maldi biotyper 3, by Bruker (1 mentions)
Bactec fx blood culture system, by BD (1 mentions)
Columbia blood agar, by BD (1 mentions)
Biotyper library v4, by Bruker (1 mentions)
Bactec plus aerobic f, by BD (1 mentions)
Vitek 2 automated system, by bioMérieux (1 mentions)
Cefotaxime, by Merck Group (1 mentions)
Cefotaxime, by Carl Roth (1 mentions)
Colistin, by Carl Roth (1 mentions)
17 protocols using maldi microflex lt
1
Peptide Synthesis and Purification
2
Peptide Synthesis and Purification
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All amino acids and resins were purchased from Novabiochem (Merck, Darmstadt, Germany). All other synthetic reagents were obtained from Novabiochem, Sigma-Aldrich (St. Louis, Missouri, USA), or Carl Roth (Karlsruhe, Germany). Peptide synthesis was performed on a MultiPep RSi peptide synthesizer (Intavis Bioanalytical Instruments AG, Köln, Germany). Peptides were purified using a 1260 Infinity II Prep HPLC system (Agilent Technologies, Santa Clara, California, USA) with a VP 250/10 NUCLEODUR 100-5 C18 ec column (Macherey-Nagel, Düren Germany) running a general gradient of 10% to 50% Acetonitrile (ACN) in H2O and validated using a 1260 Infinity I HPLC System coupled to a 6120 Quadrupole LC/MS (Agilent Technologies, Santa Clara, California, USA) with an EC 250/4 NUCLEODUR 100-5 C18 ec column (Macherey-Nagel) also running a general gradient of 10% to 50% ACN in H2O and a Microflex LT MALDI (Bruker, Bremen, Germany).
3
Detecting ESBL/AmpC E. coli from Livestock
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Extended-spectrum β-lactamases/AmpC-producing E. coli strains derived from the strain collection of the Institute for Animal Hygiene and Environmental Health, Freie Universitaet Berlin. The isolates were obtained from 7 different pig farms (n = 104) and 22 turkey farms (n = 51) in Germany. Each isolate originated from an individual sample including feces, boot swabs, manure, air or dust. ESBL/AmpC E. coli were selected using MacConkey agar (Oxoid, CM 0115, Wesel, Germany) supplemented with 1 mg/L cefotaxime, followed by species confirmation using MALDI-TOF identification (MALDI Microflex LT and Biotyper database, Bruker Daltonics, Bremen, Germany). The presence of the β-lactamase genes blaCTX, blaTEM, blaSHV and the CIT-type AmpCs (e.g., CMY-2) was confirmed by real-time PCR as described by Roschanski et al. (2014) (link).
4
Rapid Bacterial Identification and Antibiotic Resistance Profiling
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Samples were initially processed on chromogenic agar (CHROMagar Orientation, Mast Diagnostica, Reinfeld, Germany) for a rapid identification of E. coli colonies. Confirmation of single bacterial E. coli colonies with a typical morphology and of all untypical colony morphologies was performed using MALDI- TOF (MALDI Microflex LT® and Biotyper database® Bruker Daltonics, Bremen, Germany). Phenotypic antimicrobial resistance analysis of randomly picked colonies of the cecum samples obtained in necropsy was performed by using VITEK 2 system to confirm their identity as the inoculated ESBL- and AmpC- strains.
5
Isolation and Characterization of Novel Bacillus Strain
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This study was approved by the Institutional Animal Care and Use Committee of Livestock Research Institute (permit no. LRI IACUC110-35). A fecal sample obtained from a KHAPS black pig (Sus scrofa) was collected from the Kaohsiung Animal Propagation Station in Pingtung County (approximate geographic coordinates: 22.63424° N 20.60237° E), Taiwan, in 2021. Wet-weight feces (1 g) were suspended in 10 mL of phosphate-buffered saline, and the suspension was subsequently homogenized. Serial dilutions were plated on blood agar that contained 5% sheep blood for 48 h of aerobic incubation at 37 °C. All of the isolates were subjected for strain dereplication using a MALDI Microflex LT mass spectrometer (Bruker Daltonics, Bremen, Germany), as previously described [27 (link)]. One strain, named KD337-16T, could not be identified confidently. Strain KD337-16T and its phylogenetically closest reference species, including M. endophyticus BCRC 16908T, M. luteus BCRC 80739T, M. yunnanensis BCRC 80243T, M. aloeverae BCRC 80870T, and M. flavus BCRC 80069T, were routinely cultured on trypticase soy agar (TSA) at 37 °C for further taxonomic characterization, and the strains were then preserved in 10% glycerol at −80 °C.
6
Urine Culture: WASP-DTAnalysis Protocol
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Prior to automated systems, 1 μL urine specimens were cultured using the WASP®DT: Walk-Away Specimen Processor (Copan Diagnostics, Murrieta, CA) on Brilliance UTI agar (Oxoid Ltd., Basingstoke, United Kingdom). The cultures, incubated at 35°C for 18–24 h, were considered as positive if growth ≥105 CFUs/ml. We used MALDI-TOF analysis (MALDI Microflex LT, Bruker Daltonics, Bremen, Germany) to identify growing colonies. If there were three or more different types of colonies, the urine was considered as contaminated, although we classified it as negative for research purposes and it was not submitted to the identification procedure.
7
Urine Culture and Identification Workflow
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Prior to flow cytometry, the urine specimens were cultured using a WASP®DT: Walk-Away Specimen Processor (Copan Diagnostics, Murrieta, CA, USA) on Brilliance UTI agar (Oxoid Ltd., Basingstoke, United Kingdom). Cultures were incubated at 35°C for 18–24 h. Bacterial counts were expressed as the number of colony-forming units per millilitre. Growth of ≥105 CFUs/mL was considered positive. Grown colonies were identified by MALDI-TOF (MALDI Microflex LT, Bruker Daltonics, Bremen, Germany). If there were three or more types of colonies without a dominant species, the urine culture was considered as contaminated but classified as negative and not subjected to the identification procedure.
8
MALDI-TOF Identification of Microbial Isolates
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Isolate identification was performed by MALDI Microflex LT (Bruker Daltonics, Bremen, Germany) measurement, according to manufacturer formic acid extraction procedure. Briefly, in this identification method, a single colony of each strain grown overnight on YPD agar was suspended in 300µL of de-ionized water and 900µL of absolute ethanol and centrifuged at 14,462 xg for 2 min. The supernatant was discarded and the pellet was airdried. 1:1 v 70% formic acid and 100% acetonitrile were added to the pellet and vortexed. The samples were centrifuged at 14,462 xg for 2 min, and 1µLof the supernatant was spotted in duplicate onto a steel target and air-dried at room temperature. Before identification, each spot was overlaid with 1µL of HCCA (α-Cyano-4-hydroxycinnamic acid, Bruker) matrix solution saturated with organic solvent (50% acetonitrile and 2.5% trifluoroacetic acid) and air dried completely. The spectra were externally calibrated using standard ATCC Escherichia coli 25922, before plate identification. Raw spectra were processed using MALDI BIOTYPER Realtime Classification software version 3.1 (Bruker Daltonik MALDI Biotyper). Strains with score values ≥ 2 were indicated as reliable species identification.
9
MALDI-TOF MS Protein Profiling
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A standard ethanol/formic acid extraction protocol was performed by following the method described by Huang et al. (2016) . One microliter of protein extract was spotted on a 96-spot polished steel MALDI target plate. After the spots were dried at room temperature, they were overlaid with 1 µL of α-cyano-4-hydroxycinnamic acid matrix for sample/matrix cocrystallization. Mass data acquisition was performed using a MALDI-Microflex LT instrument spectrometer (Bruker Daltonics GmbH, Bremen, Germany) in positive linear mode (mass range: 2,000-20,000 Da). The bacterial test standard was used for instrument calibration. Moreover, the positive and negative controls were conducted using Escherichia coli BCRC 11634 and noninoculated matrix solution, respectively.
10
MALDI-TOF Peptide Mass Fingerprinting
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The instrument used was MALDI Microflex LT, Bruker Daltonics, Bremen, Germany. Peptide mass fingerprint product ion spectra were acquired in a linear positive mode at laser frequency of 60 Hz within a mass range of 2,000 to 20,000 daltons. Instrument parameter settings were as follows. Ion source I at 20 kV, ion source II at 18 kV, lens at 6 kV, extraction delay time of 120 ns, initial laser power of 50%, maximal laser power of 60%, and laser attenuation offset of 25% (range of 19%). For each spectrum, 240 laser shots in 40 shot steps from different positions of the target spot (random walk movement) were automatically acquired with AutoXecute acquisition control software (Flex control version 3.0; Bruker Daltonics, Leipzig, Germany).
Main spectra projection (MSP) creation was performed with a total of 68 spectra acquired for each isolate.
Main spectra projection (MSP) creation was performed with a total of 68 spectra acquired for each isolate.
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