Analysis of pineapple [ Ananas comosus (L.) Merr.] fruit proteinases by 2-D zymography and direct identification of the major zymographic spots by mass spectrometry

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Cysteine proteinases present in pineapple plants are phytotherapeutical agents with anti-edematous, anti-inflammatory, anti-thrombotic and fibrinolytic activities. Active components involved have been only partially identified as bromelain, the major proteinase in pineapple fruits. In this work, pineapple fruit extracts were analysed by 2-D zymography. Clear spots, corresponding to enzymatic activities, were excised, digested with trypsin and submitted to MALDI-ToF mass spectrometry for enzyme identification. The most representative enzymes were identified as bromelains, including their isoforms and their post-translational modifications. The novelty of the present study is the identification of proteolytic activities by means of direct MALDI-ToF MS analysis of the zymographic spots. Enzymes were identified without the need for 2-D gel electrophoresis or purification. 2-D zymography can offer not only the complete map of the enzymes present in a biological sample or in a food matrix, but it can allow also their direct identification including their isotypes.