Ultraflex maldi tof tof instrument

A 1 μL sample and 1 μL of 10 mg/mL norharmane matrix in 50% acetonitrile was applied to a MTP 384 target plate ground steel BC (Bruker Daltonics GmbH, Bremen, Germany), mixed on the target and dried under a stream of air. The samples were analyzed with an Ultraflex MALDI-TOF/TOF instrument (Bruker Daltonics GmbH, Bremen, Germany). The instrument was operated in negative acquisition mode and controlled by the FlexControl 3.3 software package. All spectra were obtained using the linear mode with an acceleration voltage of 20 kV, and pulsed ion extraction of 10 ns. The acquisition range used was from m/z 400 to 2500. Post-source decay (PSD) spectra using the Bruker Daltonics LIFT system were recorded at 8 kV precursor ion acceleration voltage and fragment acceleration (LIFT voltage 19 kV). The reflector voltage 1 and 2 were set to 29.6 and 13.9 kV, respectively.

Two µL of a 9 mg/mL solution of 2,5-dihydroxybenzoic acid in 30% (v/v) acetonitrile was applied to an MTP 384 target plate ground steel TF (Bruker Daltonics). One µL of the sample was then mixed into the DHB droplet followed by drying under a stream of hot air. The samples were analyzed with an Ultraflex MALDI-ToF/ToF instrument (Bruker Daltonics GmbH, Bremen, Germany) with a Nitrogen 337 nm laser beam. The instrument was operated in positive acquisition mode and controlled by the FlexControl 3.3 software package. The acquisition range used was from m/z 200 to 7,000. The data were collected from averaging 250 laser shots, with the lowest laser energy necessary to obtain sufficient signal to noise ratios. Peak lists were generated from the MS spectra using Bruker FlexAnalysis software (Version 3.4).

LPMO reaction products were also analyzed using MALDI-TOF mass spectrometry on an Ultraflex MALDI-ToF/ToF instrument (Bruker Daltonics, Bremen, Germany) equipped with nitrogen 337 nm laser beam. Samples (0.8 μL) were mixed with 2,5-dihydroxybenzoic acid matrix (1.6 μL, 10 mg/mL in 30% acetonitrile and 0.1% TFA), applied to a MTP 384 ground steel target plate TF (Bruker Daltonics), and air-dried. Data were collected using Bruker’s flexControl software and the spectra were analyzed using Bruker’s flexAnalysis software.

Imaging spectra were acquired on an UltraFlex MALDI-TOF/TOF instrument (Bruker Daltonics, Billerica, MA, USA) in linear positive mode with a standard 337-nm nitrogen laser operated at 100 Hz. The MALDI IMS acquisition was calibrated initially by using a mixture of standard calibrating proteins: insulin (MW 5,777.6 Da), cytochrome c (MW 12,360.1 Da), and apomyoglobin (MW 16,951.6 Da). Around 200 points in each tissue slice were scanned by 500 consecutive laser shots per point. The flexAnalysis (Bruker Daltonics, Billerica, MA, USA) recorded an average mass spectrum, which mostly presented from 3,000 to 20,000 m/z, with its coordinates on the slice. The flexImaging software (Bruker Daltonics, Billerica, MA, USA) was used for image reconstruction from acquired data.
We used the ClinProTools (CPT) to perform baseline subtraction, normalization, peak calculation, spectral alignment and statistical analyses of data processed by the flexAnalysis. Statistical significance of the peak was analyzed with a Student’s t-test or an ANOVA test on significance levels of 0.05. The CPT reported the Peak statistics, which included the mass, the mass standard deviation (Std), the averaged intensity (Ave), the P value of the t-test/ANOVA (PTTA)⁴⁶ of the individual peak derived from tumor specimen (T) or matched normal tissue (N).

To validate the LPMO activity predicted for the PHEC27213 protein, chitin, and cellulose degradation assays were performed (Hemsworth et al., 2014). In these assays, 1 μM purified PHEC27213 was added to a reaction mixture containing 2 mg/ml colloidal chitin, hepta‐N‐acetylchitoheptaose (DP7, m/z = 1,440.36) or cellulose, and 1 mM ascorbic acid as a reducing agent in 0.1 M sodium phosphate buffer (pH 7). The reactions were incubated overnight at 37 °C with shaking at 800 rpm in a thermomixer (Eppendorf). After incubation, the samples were centrifuged at 8,000 × g for 5 min at 4 °C. The oligosaccharides resulting from the enzymatic reactions were analysed by MALDI‐TOF‐MS using an Ultraflex MALDI‐TOF/TOF instrument (Bruker Daltonics GmbH) with a nitrogen 337 nm laser beam as described by Vaaje‐Kolstad et al. (2010). Penta‐N‐acetylchitopentaose (DP5, m/z = 1,033.98) was used as control of native unoxidized chitooligosaccharide. Oligosaccharides were detected according to the product masses of the resulting peaks previously described by Vaaje‐Kolstad et al. (2010) and Hemsworth et al. (2015).

MALDI-TOF analyses were conducted using an Ultraflex MALDI-TOF/TOF instrument (Bruker Daltonics, Germany) equipped with a 337-nm-wavelength nitrogen laser. All measurements were performed in positive mode. Data were collected from 100 laser shots using the lowest energy level necessary to obtain sufficient spectral intensity. The mass spectrometer was calibrated with a mixture of manno-oligosaccharides or xylo-oligosaccharides (DP, 2 to 6) obtained from Megazyme. For sample preparation, 1 μl of sample solution was mixed with 2 μl of matrix (9% 2,5-dihydroxybenzoic acid [DHB]–30% acetonitrile [vol/vol]), directly applied on a MTP 384 target plate (Bruker Daltonics, Germany), and dried under a stream of warm air.