Chan, Leo L1, 2, *Hodgkiss, I. John2 and *Lo, Samuel CL1, 1Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University; 2Department of Ecology and Biodiversity, The University of Hong Kong, Hong Kong Special Administrative Region, People's Republic of China
Keywords : Two-dimensional polyacrylamide gel electrophoresis; proteome; HAB species identification; MALDI-TOF; N-terminal amino acid sequencing
Abstract
This work had two objectives : Firstly, to document the global protein expression (proteome) of pure laboratory grown diatom and dinoflagellate cultures using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) under resting condition. Secondly, to determine whether 2-D PAGE can be used as a simple and fast method for algal identification. In this study, water soluble proteins of 7 harmful algal bloom species - Pseudonitzchia pungens, Scippsiella trochoidea, Prorocentrum triestinum, Prorocentrum micans, Prorocentrum minimum, Prorocentrum sigmoides and an unknown species of Prorocentrum (gifts from Dr. Lu Songhui), were analyzed by 2D-PAGE. With the use of immobilized-pH-gradient strips, the first dimension (isoelectric focusing) was carried out in the 3-10 pH range. The second dimension was 12.5 % SDS-PAGE. Protein visualization was performed with silver stain. In the 2D-PAGE electrophoreto-grams obtained, most of the major proteins seen fell in the pH 3.5-6.9 range. Proteomes of these 7 algal species were obtained. Identification of each protein of interest in the proteome is currently in progress using N-terminal Edman amino acid sequencing and matrix-assisted laser-desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry. When comparing 2D-PAGE chromatograms obtained using different algal species, there are proteins that are unique in each algal species. These algal species-specific proteins were mostly in the acidic pH range (pI<5.2; MW<16kDa). Our results showed that protein expression of different algal species on 2D-PAGE gels could be used for identification purposes. To conclude, 2D-PAGE allowed detailed characterization of both the isoelectric points and molecular weights of the major water-soluble proteins and it distinguished between even closely related species. With the help of other proteomic tools such as amino acid sequencing and MALDI-TOF mass spectrometry, the differentially expressed proteins in the algal proteomes could be identified and characterized.
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