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 cya nobacterium Synechocystis 6803, together with their detection and determina tion 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 apparatu s of cyanobacterium, by simply breaking cells in low-ionic-strength buffer, and subsequent injection of the total mixture of phycobilisomes into a C-4 reversed-phase column. Identification of proteins was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the sample s collected from HPLC or by measuring the protein molecular mass coupling H PLC with mass spectrometry. The latter method allows the simultaneous separ ation of the phycobiliproteins, phycocyanin and allophycocyanin, from linke r proteins and their identification, which due to their similar amino acid sequence and their similar hydrophobicity, might not be detected by denatur ing SDS-PAGE. Under the experimental conditions used, the pigment phycobili n is not removed from the polypeptide backbone, determining the hydrophobic ity of the phycoproteins and hence their interaction with the reversed-phas e 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 maint aining the unusual tertiary structure of these proteins. (C) 2001 Elsevier Science B.V. All rights reserved.