Maldi tof tof ultraflex 3 spectrometer

Mass analysis of the purified ASR1 proteins was performed using a MALDI-TOF-TOF Bruker Ultraflex III spectrometer (Bruker Daltonics, Wissembourg, France) controlled by the Flexcontrol 3.0 package (Build 51). This instrument was used at a maximum accelerating potential of 25 kV and was operated in the linear mode with m/z range from 600 to 3500. Samples (1 µL containing 15 pmol) were mixed with an equal volume of α-Cyano-4-hydroxycinnamic acid matrix solution, spotted on the target, then dried at room temperature for 10 min.
Mass spectral analysis of the protein fragments, as obtained upon thermolysin limited digestion of the purified HvASR1 and TtASR1 proteins, was performed as follows. After SDS-PAGE separation, the bands of interest were excised from the gel. The bands were then digested with trypsin (0.25 μg trypsin per μg of protein substrate). For each protein band, mass analyses were performed on a MALDI-TOF-TOF Bruker Ultraflex III spectrometer as described above, except that the instrument was operated in reflector mode. The mass standards were either autolytic tryptic peptides used as internal standards or peptide standards (Bruker Daltonics). Following MS analysis, MS/MS analyses were performed on the most intense peaks to identify the amino acid sequence of the protein band.

Purified Ag85C_Mabs recombinant protein (14 μM– 100 μg) was further incubated for 1 h in its native form with iBpPPOX, using an enzyme/inhibitor molar ratio E/I = 1:100 to ensure total inhibition. Samples of the resulting Ag85C_Mabs-iBpPPOX complex were analysed on a MALDI-TOF-TOF Bruker Ultraflex III spectrometer (Bruker Daltonics, Wissembourg, France) controlled by the Flexcontrol 3.0 package (Build 51), as described previously [24 (link)] (see S1 Appendix for full details). The total mass of the untreated protein (theoretical Mw = 32,057.83 Da; experimental Mw = 32,048.7 Da) is corresponding to the native enzyme lacking the 36 first N-terminal amino acids (i.e., M¹SVRVKARRVLSALLAAFVMPVSMAAAMTINPATAH³⁶) consisting of a Sec signal peptide cleaved at the Ala-X-Ala (i.e., A³⁵-H³⁶-A³⁷) site, as confirmed by N-terminal Edman sequencing [26 ].