5800 maldi tof ms

MALDI–TOF–MS analysis of the oligosaccharide products released by the LPMO reaction was carried out on a 5800 MALDI–TOF–MS (AB SCIEX) using 5-chloro-2-mercapto-benzothiazole (CMBT) and 2,5-dihydroxybenzoic acid (DHB) as the matrix as previously described [43 (link)]. The MS data acquisition mass range was from m/z 500 to 2500.

Freeze-dried plant tissue was first ground to a powder using a GenoGrinder (SPEX SamplePrep) before total peptides were extracted using a 50% (v/v) acetonitrile, 1% (v/v) formic acid solution at a ratio of 50 μl solvent per mg of dry mass. After overnight incubation with gentle agitation, insoluble material was pelleted by centrifugation, and soluble peptide containing supernatant was retained. For relative peptide quantification, 10 μl of the supernatant (representing 0.5 mg dry mass) was mixed with 10 μl of an unrelated control peptide (GCCSDPRCNYDHPEICGGAAGN) and 80 μl of an 80% (v/v) acetonitrile, 1% (v/v) formic acid solution. The spiked peptide extracts were mixed 1:1 with α-cyano-4-hydroxycinnamic acid [5 mg ml⁻¹ in 50% (v/v) acetonitrile / 0.1% trifluoroacetic acid (TFA) / 5 mM (NH₄ H₂PO₄)] solution before being spotted onto a MALDI plate. MALDI-TOF-MS spectra data were acquired using a 5800 MALDI-TOF-MS (AB SCIEX) operating in reflector positive mode. For relative yield determination, the isotope cluster corresponding to the transgene-derived peptide was normalised to that obtained for the internally spiked peptide control.

The lyophilized crude venom was dissolved in pure water, filtered and loaded onto a Sepax Bio-C18 column (21.2 × 250 mm, 10 μm). The HPLC was performed on a Waters 2535 system, equipped with a manual injector and two-solvent system: (A) acetonitrile with 0.1% TFA and (B) water with 0.1% TFA [18 ]. The effluent fractions corresponding to chromatographic peaks were manually collected in tubes, and lyophilized for subsequent testing. Four purified substances were collected in this way, which we designated as VMS1, VMS2, VMS3, and VMS4. Mass measurements of the isolates were performed on a matrix-assisted laser desorption/ionization time of flight mass spectrometry (5800 MALDI-TOF MS, AB Sciex, Framingham, MA, USA). The reflection method with positive ion mode was used to test the molecular mass of the samples. Tandem mass spectrometry (MS/MS) and amino acid determination by automatic amino acid analyzer, together with Edman degradation, were used to analyze the sequences of the purified components.