Mitochondria were isolated and purified from the phosphoenolpyruvate carboxykinase (PCK)- CAM plant, Ananas comosus and the malic enzyme (ME)-CAM plant, Kalanchoë pinnata sampled in daytime to identify differences in mitochondrial protein profiles between these two species. The samples were extracted in lysis buffer containing protease inhibitor mix, followed by precipitation with ice-cold acetone. Two-dimensional electrophoresis (2D-GE), coupled with matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to investigate and identify mitochondrial protein expression between these two CAM species. This investigation focused on proteins in the pI range of 3-10 pI and Mr range from 14.4 to 97.4 kDa. Staining of 2D-GE gels with Sypro Ruby revealed 389 and 415 major protein spots from mitochondria of A. comosus and K. pinnata, respectively. From these proteins, 42 major spots were selected and analyzed by MALDI-TOF MS. Peptide mass spec was used to search the MASCO database which permitted identification of 12 and 15 proteins in mitochondrial 2D-GE gels of A. comosus and K. pinnata, respectively. Mitochondria-specific proteins of A. comosus included three isoforms of ATP synthase sub unit beta, an ATP synthase subunit alpha, a phenylalanine ammonia-lyase 2, a mitochondrial ribosome protein S3, a glucose-6-phosphate isomerase, and a 14-3-3-like protein GF 14-F. Major proteins specific to K. pinnata mitochondria included ADP-ATP carrier protein, an ATP catalytic subunit A, a malate dehydrogenase, and an outer mitochondrial membrane protein (porin 2). The physiological roles of the main proteins involved in energy synthesis and mitochondrial metabolism are discussed comparatively between A. comosus and K. pinnata.
