Ultraflextreme maldi tof mass spectrometer

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)}.

Recombinant USP14 protein was desalted to remove interfering reducing agent on 30 kDa cut-off spin columns (Merck) and resuspended in water. For MALDI-TOF analysis of compound binding, 10 µg USP14 protein was incubated with 10 µM of selected compounds for 45 minutes at room temperature. Samples were then desalted to remove unbound compound on 30 kDa cut-off spin columns and resuspended in water. Approximately 0.25 µg sample was loaded onto polished stainless steel MALDI-TOF plates with sinapic acid (3,5-dimethoxy-4-hydroxycinnamic acid, Sigma) as matrix substance and air-dried until completely dry. Whole protein mass spectra were acquired on an UltrafleXtreme MALDI-TOF mass spectrometer (Bruker Daltonics) instrument operated in the linear positive ion mode with flexControl software (Version 3.4, Bruker Daltonics). Each spectrum was the accumulation of approx. 5000 laser shots for the sample spots. The MS spectra were externally calibrated using the Protein Calibration Standard II mixture (Bruker Daltonics). The MS spectra obtained were analyzed using flexAnalysis software (Version 3.4, Bruker Daltonik).

Following the protocol of Djalali Farahani-Kofoet et al. [31 (link)] for the MALDI-TOF analysis, 1 µL of the protein extract was spotted on a polished steel target (Bruker Daltonik, Bremen, Germany) and allowed to dry. Then, 1 µL of the saturated HCCA (α-cyano-4-hydroxycinnamic acid solution) was added as matrix and allowed to dry. The MALDI method was calibrated using a bacterial test standard (Bruker Daltonik). The MALDI target plate containing the protein extracts was read with an ultrafleXtreme MALDI-TOF mass spectrometer (Bruker Daltonik) working in linear positive mode and acquiring mass spectra in the range of m/z 200–20,000. Measurements were performed by flexControl v3.4 software (Bruker Daltonik). The protein spectra served as the fingerprint of the test fungi and were compared with the reference database (1832 entries) of the Bruker Filamentous Fungi Library, including 13 entries for Trichoderma sp. The MALDI Biotyper v3.1 software (Bruker Daltonik) was used to process the raw spectra, generate peak lists and perform a database search for isolate identification. A dendrogram based on PCA clustering of m/z values was constructed using the hierarchical method, correlation distance measure, and average linkage algorithm.

MALDI-TOF mass spectra
of polyols were recorded using a Bruker UltrafleXtreme MALDI-TOF mass
spectrometer (Bruker Daltonics, Germany). Polyol samples were dissolved
in THF at a concentration of 10 mg mL^–1. The solutions
thus prepared were mixed with a matrix solution of 2,5-dihydroxybenzoic
acid in THF (c = 30 mg mL^–1) and
a solution of sodium trifluoroacetate in THF (c =
10 mg mL^–1) in a volume ratio of 1:10:3. A 0.4 μL
of the mixture was spotted onto a target plate using a dried-droplet
method. The mass spectra of the samples were recorded in a reflective
positive ion mode. Calibration was performed externally using a mixture
of PMMA standards dissolved in THF and covering the measured molecular
weight range. The standard mixture was prepared according to the same
procedure as for the polyol samples and spotted to the nearest-neighbor
positions.

Peptide mixtures of each gel spot were dissolved in 0.1% TFA, desalted, and concentrated. The samples were then mixed with the same volume of matrix (CHCA in 30% ACN/0.1% TFA), spotted on a target disk, and allowed to air-dry. Samples were analyzed using a Bruker ultrafleXtreme MALDI-TOF mass spectrometer (Bruker Daltonics, Germany). The protein database search was performed using the MASCOT search engine (http://www.matrixscience.com/)27 (link). Mass tolerance was allowed within 150 parts/million (ppm). Proteins matching more than five peptides and with a MASCOT score higher than 60 were considered significant (p < 0.05).

The samples were mixed with the MALDI matrix (75 mg^.ml^-1 3-hydroxypicolinic acid in water: acetonitrile 1:1, v/v mixture) and the mixture was applied onto a ground steel MALDI target. The analyses were performed with an Ultraflextreme MALDI-ToF mass spectrometer (Bruker Daltonics) in positive and in negative ion modes. The mass spectra were calibrated externally by using synthetic peptide standards and alpha-cyano-4-hydroxycinnamic acid, used also as the MALDI matrix for calibration runs. Better detection of the covalent oligomers was obtained in the negative ion detection mode.

The matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy was performed to analyze the MW distribution of the samples using a Bruker ultraflextreme MALDI-TOF mass spectrometer (Bruker, Billerica, USA) with a 2 kHz SmartBeam laser for desorption and ionization of the samples. α-Cyano-4-hydroxycinnamic acid was used as the matrix. The original sample was diluted to 5 pmol for the measurements.