Microflex maldi tof mass spectrometer

Lipid A structures were assessed by negative- and positive-ion matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Lyophilized lipid A was extracted in chloroform-methanol, and then 1 μl was mixed with 1 μl of norharmane MALDI matrix. All MALDI-TOF MS experiments were performed using a Bruker Microflex MALDI-TOF mass spectrometer (Bruker Daltonics, Billerica, MA). Each spectrum was an average of 300 shots. Electrospray (ES) tuning mix (Agilent, Palo Alto, CA) was used for calibration. Data were analyzed using Bruker Daltonik flexAnalysis software.

The desalted peptides were mixed with a 1:1 (v/v) mixture of α-cyano-4-hydroxy cinnamic acid (HCCA) saturated with a solution comprising 30% methyl cyanide and 0.1% methanoic acid, applied to a polished steel plate, and subjected to mass characterization by a Microflex® MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany), which was operated in positive ion mode and reflector detector, and previously calibrated with an external standard (700–1800 Da). The data were processed by means of the FlexControl Software (Version 3.0, Bruker Daltonics GmbH).

The sample preparation and MALDI-TOF MS analysis was carried out according to the techniques described elsewhere [25] . Briefly, 5 µL of the cultures grown overnight were transferred into microcentrifuge tubes and subjected to ethanol and formic acid protein extraction. One µL aliquots of the supernatant were transferred onto the MALDI target plate and air dried at room temperature, followed by the addition of 1 µL of matrix solution, then air dried. Samples were then subjected to analysis using a Microflex MALDI-TOF mass spectrometer (Bruker Daltonik GmbH, Leipzig, Germany) equipped with a 60 Hz nitrogen laser. Spectra were recorded in the positive linear mode for the mass range of 2,000 to 20,000 Da at the maximum laser frequency. The raw spectra were then analysed using the MALDI Biotyper 3.0 software package (Bruker Daltonik GmbH, Bremen, Germany) under the default settings. Measurements were performed via the automatic mode, without any user intervention.

For matrix-assisted laser desorption ionization time of flight (MADLI-TOF) mass spectrometry measurements, peptide in solvent was mixed with a saturated solution of a-cyano-4-hydroxycinnamic acid (a-HCCA) in a mixture of 70% methanol and 0.05% trifluoroacetic acid. This solution was dried dropwise onto an MSP AnchorChip target plate (Bruker, Billerica, MA). Mass spectra were collected on a Bruker Microflex MALDI-TOF mass spectrometer, calibrated with the Bruker Peptide Calibration Standard II (Billerica, MA). Spectra were analyzed using FlexAnalysis software (Bruker, Billerica, MA).

¹H and ¹³C NMR spectra were collected on a 400 MHz NMR spectrometer which operated at 400 MHz for ¹H and 100 MHz for ¹³C (Bruker Company). Mass spectra were recorded on a Microflex MALDI-TOF mass spectrometer (Bruker Daltonics). High-resolution mass spectra were obtained from a triple quadrupole GC/MS (Agilent Technologies). Elemental analysis was obtained from a CHNS/O analyzer (Flash 2000, Thermo Scientific). Absorption spectra were obtained from a UV-2250 UV-Vis Spectrophotometer (SHIMADZU, Japan) and emission spectra were obtained from a Carry Eclipse Fluorescence Spectrophotometer (Agilent Technologies). The absolute fluorescence quantum yields and lifetimes were determined using FLS980 Spectrometer (Edinburgh Instruments). The pH buffers were measured from an Ohaus pH meter. Amounts of Hg(ii) ion in water samples were verified by an inductively coupled plasma-optical emission spectrometer (ICP-OES) (iCAP 6500, Thermo Scientific).

Reagents Rink amide resin, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt))-OH, Dicyclohexylcarbodiimide (DCC), and 1-Hydroxy-6-chlorobenzotriazole (6-Cl-HOBt) were purchased from AAPPTec (Louisville, KY, USA). Reagents acetonitrile (ACN), trifluoroacetic acid (TFA), dichloromethane (DCM), diisopropylethylamine (DIPEA), N,N-dimethylformamide (DMF), ethanedithiol (EDT), isopropanol (IPA), methanol, and triisopropylsilane (TIS) were acquired from Merck (Darmstadt, Germany). SPE Supelclean™ LC-18 columns were purchased from Sigma-Aldrich (St. Louis, MO, USA). Trypticase Soy Agar (TSA), Mueller Hinton Agar (MHA), and Plate Count Agar (PCA) media were purchased from Scharlau. Mueller Hinton Broth medium was purchased from Merck. The Escherichia coli ATCC 25922 bacterial strain was purchased from the ATCC. The MCF-7 and HeLa cell lines were obtained from ATCC^® (Manassas, VA, USA). All the reagents were used directly, without previous purification.
¹H spectra were recorded at 400 MHz on a Bruker Advance 400 instrument. RP-HPLC analyzes were performed on a Chomolith C18 column (Merck, Kenilworth, NJ, USA, 50 mm), using an Agilent 1200 Liquid Chromatograph (Agilent, Omaha, NE, USA). The products were analyzed on a Bruker Impact 2 LC Q-TOF MS equipped with electrospray ionization (ESI) in positive mode and on a Bruker MicroFlex MALDI-TOF mass spectrometer.