Ultraflex 3 tof tof

Transglycosylase reactions were carried out using NAG6 as substrate with 0.032 mg mL⁻¹ of the corresponding chitinase variant and 2.5 mg mL⁻¹ substrate in 100 mM phosphate potassium at pH 5.5 (total volume, 0.5 mL). Reactions were incubated at 900 rpm in a thermo shaker for 24 h at 45 °C as described above and then stopped by adding 0.2 M NaOH.
The molecular weight of produced the COS was assessed by MALDI-TOF-MS using a mass spectrometer with Ultraflex III TOF/TOF (Bruker, Billerica, MA, USA) and an NdYAG laser. Registers were taken in positive reflector mode within a mass interval of 40−5000 Da, external calibration and 20 mg mL⁻¹ 2,5-dihydroxybenzoic in acetonitrile (3:7) as a matrix. Samples were mixed with the matrix in a 4:1 ratio, and 0.5 μL was analyzed. The amounts of NAG oligomers were deduced based on the MS peak areas generated by each oligomer, and the percentage of their relative abundance referred to the total peak areas detected for all NAG oligomers detected in the reaction.

The mass detectors currently used chromatographic systems (liquid and mass), have a detection limit of approximately 2000 Da and become a limiting factor when working with phlorotannins, which are known to be larger. Then, for the analysis of oligomers, above 500 Da in the extracts was carried out with a matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI–TOF-MS). MALDI–TOF-MS spectra were recorded for samples PWE, PD20 and PD2 to assess the range of mass fragmentation. The equipment used was Bruker Ultraflex III TOF/TOF equipped with a N₂ laser of 337 nm, 1 µm of laser beam diameter and operated in positive mode. MALDI–TOF-MS spectra data treatment was carried out with data-analysis software (Bruker, Billerica, MA, USA). MALDI–TOF-MS analysis was performed by following a protocol that was previously proposed [10 (link)].

Cyclization of linear target peptides was monitored by MALDI MS. The reaction mixture (10–20 μl) was desalted using C18 zip tips and eluted in 4 μl 50% acetonitrile, 0.1% trifluoroacetic acid (TFA). A saturated MALDI matrix solution (α–cyano-4-hyroxycinnamic acid) prepared in 95% acetonitrile, 0.1% TFA was diluted 1:22 such that the final matrix solution comprised 90% acetonitrile, 0.1% TFA and 1 mM NH₄H₂PO₄. Eluted samples were mixed 1:4 with the MALDI matrix, spotted onto a MALDI plate and analysed by an Ultraflex III TOF/TOF (Bruker) in positive reflector mode.

For preparation of diethylene triamine pentaacetic acid (DTPA) labeled-sulfonamide (ATS-DTPA), ATS and DTPA dianhydride (w/w 1:50) was incubated at 25°C with bicarbonate buffer (pH 8.5) for 4 hours. Then, the conjugated-compounds were purified by high pressure liquid chromatography with G-10 column. For molecular weight assay, liquid chromatography-mass spectrometry (LTQ Orbitrap XL™ Hybrid Ion Trap-Orbitrap, Thermo Scientific, USA) was performed for ATS analysis. And matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UltraflexIII TOF/TOF, Bruker Daltonics GmbH, Germany) was performed for DTPA and ATS-DTPA detection. Briefly, the compounds were crystallized with an equal volume of freshly prepared α-cyano-4-hydroxycinnamic acid matrix (10 mg/ml in 50% acetonitrile/0.5% TFA) for mass spectrometry analysis. The calibration standards were used as external calibrator with mass accuracy within 100 ppm. The spectra were processed using FlexAnalysis™ 3.0 software (Bruker, Germany). The ATS-DTPA conjugation efficiency with indium-111 was evaluated from the radiolabeling yields of ATS-DTPA-¹¹¹In determined by instant thin layer chromatography (ITLC) (AR-2000 radio-TLC Imaging Scanner, Bioscan, France) on silica gel impregnated glass fiber sheets (PALL corporation, USA) using PBS buffer, pH 7.4, as the mobile phase.

The extracted peptides from the 32 reference strains of S. suis were mixed with the sinapinic matrix solution [sinapinic acid in 5% (v/v) TFA in absolute ACN] and then spotted onto the MALDI target plate and allowed to crystalize at room temperature before inserting the MALDI target plate into the MALDI-TOF-MS instrument. The mass spectrometry (MS) spectra were collected using an Ultraflex III TOF/TOF (Bruker Daltonik, GmbH) instrument in a linear positive mode with a mass range between 2–20 kDa. Five hundred shots were reiterated and accumulated with a 50 Hz laser per SS. Likewise, this MALDI-TOF-MS procedure was repeated for all 32 serotypes of S. suis. All MS spectra were analyzed for fingerprint spectra and subjected to PCA using the FlexAnalysis version 3.4 and ClinProTool version 3.0 software (Bruker Daltonik, GmbH). The ACTH fragment 18–39 (human), insulin oxidized B chain (bovine), insulin (bovine), cytochrome C (equine), and apomyoglobin (equine) were used as the external protein calibrations. Analysis of variance (ANOVA), Student’s t-test and statistics in the software, were used to assess the statistically significant differences in the PMFs across bacterial strains.

The extracts of the duct sections were analysed using an Ultraflex III TOFTOF (time-of-flight) mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a 200 Hz all-solid-state laser (SmartBeam) and controlled by the FlexControl 2.4 software package using a previously reported method for cone snail venom analysis⁴ . To analyse peptides with a mass range of 1000 Da and 10,000 Da, Ultraflex III was operated in both linear-positive and reflectron-positive mode using CHCA as a matrix. Spectra calibration was performed externally using a peptide calibration mixture (206195, Bruker Daltonics, Bremen, Germany). A CHCA solution was made by the dilution of acetone saturated with CHCA 1 in 10 with an acetone:acetonitrile:water (6:3:1) solution. The raw samples were diluted 1 in 100 with 0.1% TFA, and 2 ml of diluted matrix solution mixed with 1 ml sample and spotted onto a polished steel target. For all samples, 400 shots were acquired using a random walk function at a laser frequency of 200 Hz and saved, with 10 replicates of each sample averaged⁴ . Data were loaded into Clinprot Tools (Bruker Daltonics, Bremen, Germany) to visualize the 8 individual duct sections and injected predatory and defensive venoms in ‘gel view’ using a colorimetric gradient to show the abundance of the components in respective fractions.