IssA activity; MALDI-TOF MS
Description
MALDI-TOF MS-based protease assays. Each reaction consists of 1mM substrate peptide and 1 micromolar IssA protease (IMG GeneID 2558297680; purified from heterologous Pichia pastoris). Reaction buffer was 10 mM sodium acetate, pH 5.2. figure 5a = canonical flg22 as substrate (QRLSTGSRINSAKDDAAGLQIA) figure 5b = Dyella japonica MF79 flg22 as substrate (ERLSSGMRINSAKDDAAGLAIS) figure 5c = AtPEP1 from Arabidopsis thaliana as substrate (ATKVKAKQRGKEKVSSGRPGQHN) figure 6s= canonical flg22 substrate as in figure 5a, but IssA was either inactivated by treatment with PMSF or mock treated.
Files
Steps to reproduce
Substrate peptides were diluted in 10 mM sodium acetate, pH 5.2 at 1 mM. IssA protease was added (1 micromolar) to initiate protease reactions. Time points were taken by mixing equal volumes of reaction with matrix (saturated 2,5-dihydroxybenzoic acid in acetonitrile) and immediately spotting onto a target. Samples were analyzed using a Bruker-Daltonics Microflex LRF with a pulsed N2 laser with reflectron acquisition (3000 shotes, 337 Hz, 60 Hz pulse).