Proteomic assessment of an established technique for carboxysome enrichment from Synechococcus PCC7942

Carboxysomes are protein-bound, polyhedral microbodies within cyanobacteria, containing the key enzyme for photosynthetic CO₂ fixation, ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco). Sequencing of cyanobacterial genomes has revealed that cyanobacteria possess one or other of two types of carboxysomes. Cyanobacteria containing form 1A Rubisco possess α-carboxysomes, while those with form 1B Rubisco possess β-carboxysomes. Given the central importance of carboxysomes in the CO ₂-concentrating mechanism of cyanobacteria, understanding the nature and composition of these structures is of considerable importance. In an effort to develop techniques for the characterization of the structure of β-carboxysomes, particularly the outer protein shell, we have undertaken a proteomic assessment of the Percoll-Mg²⁺ carboxysome enrichment technique using the freshwater cyanobacterium Synechococcus sp. PCC7942. Both matrix-assisted laser desorption-ionization - time of flight mass spectrometry (MALDI-TOF MS) and multidimensional protein identification technology (MuDPIT) methods were used to determine the protein content of a novel carboxysome-rich fraction. A total of 17 proteins were identified using MALDI-TOF MS from enriched carboxysome preparations, while 122 proteins were identified using MuDPIT analysis on the same material. The carboxysomal protein CcmM was identified by MALDI-TOF MS as two distinct proteins of 38 and 58 kDa. The only other carboxysomal proteins identified were the large and small subunits of Rubisco (RbcL and RbcS). Reasons for the lack of evidence for the expected full complement of carboxysomal proteins and future directions are discussed.