Optimization of 2D-PAGE protocols for proteomic analysis of two nonaxenic toxin-producing freshwater cyanobacteria: Cylindrospermopsis raciborskii and Raphidiopsis sp.
OBJECTIVE
To optimize a protocol for the extraction and an in-depth analysis of the soluble protein fraction of two nonaxenic toxin-producing cyanobacteria Cylindrospermopsis raciborskii (hepatotoxin-producing), and Raphidiopsis sp. (neurotoxin-producing), using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).


RESULTS
The soluble protein fractions from strains of C. raciborskii and Raphidiosis sp. with different toxicity phenotypes were analysed by 2D-PAGE. Specific protocols were optimized specifically for each strain. Between 500 and 700 sharp protein spots were distinguished in a single 4-7 pH range 2D-PAGE for each cyanobacterium. Comparison of the protein maps of C. raciborskii CS-505 (a cylindrospermopsin-producing strain) and Raphidiopsis sp. D9 (saxitoxin-producing strain) against the nontoxic C. raciborskii strain CS-509 revealed many unique proteins in each protein map. We confirmed that the resolved proteins were cyanobacterial by identifying three randomly chosen protein spots from a Raphidiopsis sp. strain D9 2D-PAGE, using high-performance liquid chromatography (HPLC) tandem mass spectrometry (MS).


CONCLUSIONS
The 2D-PAGE conditions presented here provide a robust protocol for proteomic studies in two CYN- and STX-producing model organisms, C. raciborskii and Raphidiopsis sp.


CONCLUSIONS
We present the first protocols for proteomic analyses of Cylindrospermopsis raciborskii and Raphidiopsis sp.
Plominsky
A M
AM
Laboratory of microbial ecology and environmental toxicology, Departamento de Genética Molecular y Microbiología, Pontificia Universidad Católica de Chile, Santiago, Chile.
Soto-Liebe
K
K
Vásquez
M
M
D001426:Bacterial Proteins / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D000458:Cyanobacteria / Q000737:chemistry*; D046891:Cylindrospermopsis / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005618:Fresh Water / Q000382:microbiology*; D013058:Mass Spectrometry; D020543:Proteome / Q000032:analysis*
D001426:Bacterial Proteins; D020543:Proteome