Characterization by PMF MALDI-TOF analysis of plant cysteine endopeptidases

OBREGÓN, D.; TORRES, M.J.; LÓPEZ, L.; MORCELLE, S.; BRUNO, M.; LIGGIERI, C.; TREJO, S.

Pilar, Bs. As. Argentina.

Congreso; 1st. Annual Iberoamerican Proteomics Congress; 2007

LAHUPO (Latinamerican Human Proteome Organisation)

CHARACTERIZATIO FOR PMF MALDI-TOF ANALYSIS OF PLANT CYSTEINE ENDOPEPTIDASES David Obregón, María José Torres, Laura lópez, Susana Morcelle, Constanza Liggieri, Sebastián Trejo Our group has prevously isolated characterized about twenty cysteine endopeptidses from latex and fruits. The aim of this work was to obtain and compare the peptide mass fingerprint (PMF) by MALDI TOF MS for several of those plant proteases isolated from latex of Araujia hortorum, Araujia angustifolia, Asclepias fruticosa and Carica quercifolia, and from fruits of Bromelia hieronymi. They differ in their pI values as well as their molecular weight and biochemical characteristics. Palnt proteases were separated by SDS PAGE and visualized by staining the gels according to the colloidal Coomassie method. The selected bands were cut, washed and dried under vacuum. The gel fragments were treated with a solution of 100 mM NH4HCO3 containing 10 mM DTT, centrifuged and washed with acetonitrile. Then were incubated in a solution of 100 mM NH4HCO3 containing 50 mM iodoacetamide for 20 min at room temperature in darkness. Tryptic digestion was achieved by using a porcine pancreas dimethylated trypsin, proteomic grade, (0.4 mg/ml) during 12 h at 37ºC. the resulting peptides were recovered by centrifugation, washed with milli Q water and acetonitrile, dried in a SpeedVac, redissolved in 0.1% (v/v) TFA and analysed by MALDI-TOF MS using a cyano-4-hydroxycinnamic acid (HCCA) matrix. The PMF analysis showed the enzymes have some equivalent peaks, revealing a high degree of homology among them, which would reflect the presence of conserved domains in these plant peptidases belonging to different families (Asclepiadaceae, Caricaceae and Bromeliaceae). By means of the MASCOT tool, searches were made in order to identify studied with these triptic maps. No identification was possible due to only a few plant cysteine proteinases are consigned in the available databases. The obtained results represent an interesting contribution for the databases containing PMF on plant endopeptidases.