Mtp 384 ground steel target plate tf

Product mixtures in reaction supernatants were qualitatively assessed using a matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) UltrafleXtreme mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany), equipped with a Nitrogen 337-nm laser. Reaction mixtures (1 µL) were applied to an MTP 384 ground steel target plate TF (Bruker Daltonics) together with 2 µL of 9 mg/mL of 2,5-dihydroxybenzoic acid (DHB) dissolved in 30% acetonitrile, followed by drying under a stream of air. Data collection and analysis were preformed using the Bruker FlexAnalysis software.
Quantification of oxidized chitobiose (GlcNAcGlcNAc1A) was achieved using a Dionex Ultimate 3000 UHPLC system (DionexCorp., Sunnyvale, CA, USA) equipped with a Rezex RFQ-Fast Acid H + (8%) 7.8 × 100 mm column (Phenomenex, Torrance, CA) operated at 85 °C. 8 µL samples were injected into the column and the reaction products were eluted isocratically, using 5 mM sulfuric acid as mobile phase, and detected via UV absorption at 194 nm. Data collection and analysis were performed with the Chromeleon 7.0 software. Standards were generated in-house by complete oxidation of N-acetyl-chitobiose (Megazyme; 95% purity) with a chitooligosaccharide oxidase from Fusarium graminearum (ChitO)^{85 (link)}, as previously described^{77 (link)}.

Two microliter of a saturated solution of 2,5- dihydroxybenzoic acid (DHB) in 30% acetonitrile was applied on an MTP 384 ground steel target plate TF (Bruker Daltonics). One microliter sample was then mixed with the DHB droplet and dried under a stream of air. The samples were analyzed with an Ultraflex MALDI-ToF/ToF instrument (Bruker Daltonics GmbH, Bremen, Germany) controlled by the FlexControl 3.3 software package. The instrument was operated in positive acquisition mode and the acquisition range used was from m/z 0 to 3000. The data were collected from averaging 250 laser shots, with the lowest laser energy necessary to obtain sufficient signal to noise ratios. Lithium doping was obtained by mixing two microliter sample with 0.5 microliter 100 mM LiCl 100 mM LiCl, one microliter of this mixture was applied on the target plate as described above.

Native and oxidized oligosaccharides were analyzed by HPAEC using a Dionex ICS-5000 system equipped with pulsed-amperometric detection (PAD) and a CarboPac PA1 analytical column with a CarboPac PA1 guard column (Dionex, Sunnyvale, CA, US). A 0.25 mL/min flow and 50-min gradient were employed as previously described [53 (link)]. Additional product analysis was performed by MALDI-TOF MS, using an Ultraflex MALDI-TOF/TOF instrument (Bruker Daltonics, Bremen, Germany) equipped with a nitrogen 337-nm laser beam, as described previously [1 (link)]. Prior to MALDI-TOF MS analysis, samples (1 μL) were spotted on an MTP 384 ground steel target plate TF (Bruker Daltonics) together with 1 μL of a saturated 2,5-dihydroxybenzoic acid solution and dried.

The oligosaccharides released by LPMO action and sequential LPMO-endoglucanase treatment were analyzed by high-performance anion-exchange chromatography (HPAEC) on a Dionex ICS3000 system equipped with pulsed amperometric detection (PAD), using a 50-min gradient (33 (link)) for cellulosic substrates and a 75-min gradient (13 (link)) for hemicellulosic substrates.
The oligosaccharides were further analyzed using MALDI-TOF MS. The analysis was carried out on an Ultraflex MALDI-TOF/TOF instrument (Bruker Daltonics, Bremen, Germany) equipped with a nitrogen 337-nm laser beam, as described earlier (5 (link)). Samples (2 μl) were applied to an MTP 384 ground steel target plate TF (Bruker Daltonics) together with 4.5 mg of 2,5-dihydroxybenzoic acid (DHB) matrix dissolved in 0.5 ml of 30% acetonitrile. Data were collected with the lowest laser energy necessary to obtain sufficient quality spectra, using Bruker's flexControl software. Spectra were analyzed using Bruker's flexAnalysis software. All samples analyzed in this study contained 50 mM sodium acetate, which suppressed the formation of potassium adducts. The signal of amperometric detection is expressed in nanocoulombs (nC).

Products in reaction supernatants were assayed qualitatively using a matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) UltrafleXtreme mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany) equipped with a Nitrogen 337-nm laser. Reaction mixtures (1 µL) were applied to an MTP 384 ground steel target plate TF (Bruker Daltonics) and mixed with 2 µL of 9 mg/ml of 2,5-dihydroxybenzoic acid dissolved in 30% acetonitrile, followed by air-drying. Data collection and analysis were carried out using the Bruker FlexAnalysis software.

Product formation from hemicellulosic substrates was assayed qualitatively using an matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) UltrafleXtreme mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany) equipped with a Nitrogen 337-nm laser. Reaction products (1 μl) were applied to an MTP 384 ground steel target plate TF (Bruker Daltonics) together with 2 μl of 9 mg/ml of 2,5-dihydroxybenzoic acid (DHB) dissolved in 30% acetonitrile, followed by air-drying. Spectra were collected using Bruker FlexControl software and analysed with Bruker flexAnalysis software.

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).