Peptide standard

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.

For ESI-MS^/MS analysis, the mass of interest (854.42) was selected using an inclusion list and fragmented using data-directed analysis (DDA) with the following parameters: top3 precursor selection (inclusion list only); MS2 threshold, 50,000; scan time, 0.5 s without dynamic exclusion. Collision energy (CE) was ramped between 15 and 20 at low mass (50 m/z) and 40 to 100 at high mass (2,000 m/z). Further increase of the CE from 20 to 30 and from 60 to 120 led to complete fragmentation.
For MALDI-TOF-MS, the samples were mixed with α-cyano-4-hydroxycinnamic acid as matrix and analyzed on an Autoflex Speed MALDI-TOF/TOF mass spectrometer (Bruker Daltonics GmbH). The instrument was controlled by a flexControl (version 3.4; Bruker) method optimized for peptide detection and calibrated using peptide standards (Bruker). For sequence analysis, fragments produced by post-source decay (PSD) were measured using the LIFT method (Bruker). All spectra were processed in flexAnalysis (version 3.4; Bruker).

Mass analysis of the purified mutated N_TAIL proteins was performed using an Autoflex II ToF/ToF (Bruker Daltonics). Spectra were acquired in a linear mode. 15 pmol of samples were mixed with an equal volume (0.7 μL) of sinapinic acid matrix solution, spotted on the target and dried at room temperature.
The identity of the purified N_TAIL proteins was confirmed by mass spectral analysis of tryptic fragments obtained by digesting (0.25 μg trypsin) 1 μg of purified recombinant protein isolated onto SDS-PAGE. The tryptic peptides were analyzed as described above and peptide fingerprints were obtained and compared with in-silico protein digest (Biotools, Bruker Daltonics). The mass standards were either autolytic peptides or peptide standards (Bruker Daltonics).