De novo sequence analysis and intact mass measurements for characterization of phycocyanin subunit isoforms from the blue-green alga Aphanizomenon flos-aquae.
In this work, partial characterization of the primary structure of phycocyanin from the cyanobacterium Aphanizomenon flos-aquae (AFA) was achieved by mass spectrometry de novo sequencing with the aid of chemical derivatization. Combining N-terminal sulfonation of tryptic peptides by 4-sulfophenyl isothiocyanate (SPITC) and MALDI-TOF/TOF analyses, facilitated the acquisition of sequence information for AFA phycocyanin subunits. In fact, SPITC-derivatized peptides underwent facile fragmentation, predominantly resulting in y-series ions in the MS/MS spectra and often exhibiting uninterrupted sequences of 20 or more amino acid residues. This strategy allowed us to carry out peptide fragment fingerprinting and de novo sequencing of several peptides belonging to both alpha- and beta-phycocyanin polypeptides, obtaining a sequence coverage of 67% and 75%, respectively. The presence of different isoforms of phycocyanin subunits was also revealed; subsequently Intact Mass Measurements (IMMs) by both MALDI- and ESI-MS supported the detection of these protein isoforms. Finally, we discuss the evolutionary importance of phycocyanin isoforms in cyanobacteria, suggesting the possible use of the phycocyanin operon for a correct taxonomic identity of this species.
Rinalducci
Sara
S
Department of Environmental Sciences, University of Tuscia, Viterbo, Italy.
Roepstorff
Peter
P
Zolla
Lello
L
D000595:Amino Acid Sequence; D046890:Aphanizomenon / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D001557:Benzenesulfonates / Q000737:chemistry; D017879:Isothiocyanates / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010798:Phycocyanin / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D020539:Sequence Analysis, Protein / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D001426:Bacterial Proteins; D001557:Benzenesulfonates; D017879:Isothiocyanates; D020033:Protein Isoforms; D010798:Phycocyanin; C015308:4-isothiocyanatobenzenesulfonate