Microflex 2 mass spectrometer

Xyloglucan oligosaccharides released after 24 h of incubation at 37 °C by 0.2 μM of Cel44O, 0.1 μM of Cel9X and 0.1 μM of the fusion 9X-44O on 3.5 g/L tamarind xyloglucan, were subjected to MALDI-TOF analyses. Similarly, samples obtained during the sequential degradation of XXXG by 30-minute incubations at 37 °C with either 1 μM of α-xylosidase or 1 μM of β-glucosidase were analyzed using the same procedure. MALDI-TOF analyses were performed on a Microflex II mass spectrometer (Bruker Daltonics) as previously described53 (link). One μL of matrix (10 mg of 2,5-Dihydroxybenzoic acid in 1 mL of CH₃CN/H₂O/50/50 (v/v), 0.1% formic acid (v/v)) was added to 1 μL of sample (100 pmoles) in the same solution. In reflectron positive mode used for acquisition (mass range 160–3000 Da), accuracy was less than 50 ppm.

Matrix-assisted laser desorption ionization mass spectra analyses were performed on a Microflex II mass spectrometer (Bruker Daltonics, MA, USA). One μL of matrix [10 mg of 2,5-Dihydroxybenzoic acid in 1 ml of CH₃CN/H₂O 50/50 (v/v), 0.1% formic acid (v/v)] was added to 1 μL of intact LPMO protein sample (100 pmol) in the same solution. Then, the mixture was allowed to dry at room temperature. Data acquisition was operated using the Flex control software. External mass calibration was carried out on Peptide calibration standard (Bruker Daltonics).

Matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) analysis were performed on a Microflex II mass spectrometer (Bruker Daltonics, Germany). Depending on the concentration, samples were used directly or concentrated following the ZipTip C4 Millipore protocol. A saturated solution of sinapinic acid made in acetonitrile-water-trifluoroacetic acid (50:50:0.1) was used as the matrix. Samples were treated according to the dry droplet method; mixtures were allowed to dry at room temperature. Deposits were re-crystallized by the addition of matrix. Data were acquired in a positive linear mode; depending on the mass analyzed, the range was set from 20 to 50 kDa or from 50 to 85 kDa, and pulsed ion extraction was fixed respectively to 350 ns or 500 ns. External mass calibration was done just before the acquisition of the sample using protein calibration standard II (Bruker Daltonics, Germany). Mass spectra were examined in Flex Analysis software, no smoothing or baseline subtraction was applied. For each version of SipB protein (wild-type or variant) and for each condition of production (with or without IacP) independent preparations of purified protein and mass spectrometry analysis were made at least twice.