Axima performance maldi tof tof mass spectrometer

E. coli expressing empty plasmid or Cfr were grown at 37°C to an OD₆₀₀ of 0.4–0.6 with shaking by diluting an overnight culture 1:100 into LB media containing ampicillin (100 µg/ml) and AHT inducer (30 ng/ml). Total RNA purification, oligo-protection of the 23S rRNA fragment C2480-C2520, and RNaseT1 digestion were performed as described previously (Stojković and Fujimori, 2015 (link); Andersen et al., 2004 (link)). Mass spectra were acquired in positive ion, reflectron mode on an AXIMA Performance MALDI TOF/TOF Mass Spectrometer (Shimadzu). Relative peak intensity values were calculated using the Shimadzu Biotech MALDI-MS software.

Fourier-transform infrared (FTIR) spectra were recorded on a Spectrum One System (PerkinElmer, Waltham, MA, USA). The KBr pellet method was applied to the analysis of nanoparticle powder by mixing the sample powder (ca. 1% of the KBr amount) with KBr. UV-Vis absorption spectra were measured on a UV-Vis spectrophotometer (Model U-3900, Hitachi, Japan). Fluorescence spectra were recorded using a Multi-Mode Microplate Reader (Model SpectraMax iD3, Molecular Devices LLC, San Jose, CA, USA).
Mass spectra were acquired using an AXIMA Performance MALDI TOF-TOF mass spectrometer (Shimadzu Corporation, Kyoto, Japan) equipped with a 337 nm nitrogen laser. Ion source, lens, and linear detector voltages were set at 20.0, 6.0, and 2.8 kV, respectively. MALDI-TOF mass spectra were acquired in the positive linear mode with 200 shots from random positions on the same sample spot. Spectra were acquired as the sum of the laser shots. The bacteria were suspended in aqueous 70% (v/v) formic acid (FA). The saturated matrix solution was prepared by dissolving α-cyano-4-hydroxycinnamic acid (CHCA) into a solution of 50% acetonitrile/water containing 0.1% formic acid. A 1 µL aliquot of bacteria sample in 70% FA mixed with an equal volume of CHCA matrix solution was spotted on a target plate and allowed to air-dry before MALDI-MS analysis.

The molecular masses of B. atrox toxins were initially estimated according to their SDS-PAGE profile, by interpolating a linear logarithmic curve of the relative molecular mass of standard proteins versus the distance of migration of sample proteins in the gel.
MALDI-TOF mass spectrometry analyses were also performed to determine the molecular mass of intact proteins, using an AXIMA Performance MALDI-TOF/TOF mass spectrometer (Shimadzu, Japan) previously calibrated with known molecular mass standards. Mass spectra were acquired in linear mode, evaluating the range from 5,000 to 50,000 m/z. The samples were diluted in 50 μL of Milli-Q water, mixed in a 1:1 ratio with a matrix consisting of sinapinic acid (10 mg/mL) in 50 % acetonitrile and 0.1 % TFA, and applied on the MALDI plate using the dried-droplet method.

The gangliosides fractions extracted from FFPE tissues were analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI TOF) mass spectrometry. MALDI mass spectra were obtained using an AXIMA Performance MALDI TOF/TOF mass spectrometer (Shimadzu Biotech) with a 337 nm N₂ laser using delayed extraction, in positive reflectron mode. The glycolipid extracts were resuspended in 10 μL of 50% methanol, from which 1 μL was mixed with an equal volume of a saturated solution of 2,5-dihydroxybenzoic acid (DHB) (Sigma-Aldrich, 85707) in 50% acetonitrile (Sigma-Aldrich, 34851) on top of the MALDI target plate. Samples were allowed to dry off at room temperature and were recrystallized through the addition of 1 μL of ethanol. Two hundred laser shots were accumulated for each mass spectrum, with a 50 Hz laser repetition rate. In this analysis, signals between m/z 1.000 and 2.000 were collected. The data was processed using MALDI-MS software from Axima Biotech Launch pad software pack.
The spectra were externally calibrated against ProteoMassTM peptide MALDI-MS calibration kit (Sigma-Aldrich, MSCAL2-1KT). The peaks corresponding to NeuAcGM3 and NeuGcGM3 were confirmed by comparison with purified samples of both gangliosides.

The samples obtained from sculptin incubation with factor Xa or thrombin were analyzed by MALDI-TOF MS, using 3,5-dimethoxy-4-hydroxycinnamic acid as the matrix on AXIMA Performance MALDI TOF/TOF Mass Spectrometer (Shimadzu, Japan)^{39 (link)}. Briefly, the matrix solution (1 µL) and sculptin digest (1 µL) of either factor Xa or thrombin were premixed in eppendorf tube and 0.5 µL of the pre-mixed sample was spotted on the steel target plate. The mass spectra were recorded by MALDI-TOF instrument (Shimadzu, Japan). A linear time-of-flight in MALDI-MS instrument was employed with vacuum 3 × 10⁻⁷ Torr and power of laser 120. The spectra were generated by averages of 50–100 automatic laser shots. The mass spectra were analyzed using Axima Performance proteomics suit. The mass tolerance was 0.2%.

MALDI-TOF MS analyses were performed in an AXIMA Performance MALDI TOF/TOF Mass Spectrometer (Shimadzu, Nakagyo-ku, Kyoto, Japan). Briefly, 0.5 µL of the sample was co-crystalized with an equal volume of sinapinic acid matrix solution and the mixture was spotted and dried on a steel target plate. Samples were analyzed in positive, linear mode under a vacuum of 3 × 10⁻⁷ Torr and laser power of 120 in a 4 kDa to 40,000 kDa window.