High-performance liquid chromatography coupled on-line with electrospray ionization mass spectrometry for the simultaneous separation and identification of the Synechocystis PCC 6803 phycobilisome proteins

The complete resolution of the protein components of phycobilisome from cyanobacterium Synechocystis 6803, together with their detection and determination of molecular mass, has successfully been obtained by the combined use of HPLC coupled on-line with electrospray ionization mass spectrometry. The method proposed consists of the isolation of the light-harvesting apparatus of cyanobacterium, by simply breaking cells in low-ionic-strength buffer, and subsequent injection of the total mixture of phycobilisomes into a C4 reversed-phase column. Identification of proteins was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the samples collected from HPLC or by measuring the protein molecular mass coupling HPLC with mass spectrometry. The latter method allows the simultaneous separation of the phycobiliproteins, phycocyanin and allophycocyanin, from linker proteins and their identification, which due to their similar amino acid sequence and their similar hydrophobicity, might not be detected by denaturing SDS-PAGE. Under the experimental conditions used, the pigment phycobilin is not removed from the polypeptide backbone, determining the hydrophobicity of the phycoproteins and hence their interaction with the reversed-phase column as well as in determining the protein-protein interaction into the phycobilisome aggregation. Removal of the pigment, in fact, abolishes HPLC separation, emphasizing the essential role that the pigments play in maintaining the unusual tertiary structure of these proteins.