Proteomic Study of Micro-Algae: Sample Preparation for Two-Dimensional Gel Electrophoresis and De Novo Peptide Sequencing Using MALDI-TOF MS
Fred Wang-Fat Lee,
Samuel Chun-Lap Lo.
Proteins are the major players in most processes of living cells and study of the proteome has great relevance to investigations on cells and organisms at the molecular level. 2-DE is a core and powerful proteomic technique to study protein expression and function in living organisms and it allows a fast overview of changes that occurred in cells at the proteome level. Although data on DNA sequences from large scale genomic sequencing projects has greatly facilitated the identification and characterization of proteins, genomic sequences of many organisms, including that of many microalgae are still unknown. Thus, de novo protein/peptide sequencing techniques are important for acquiring amino acid sequences of proteins from organisms with incomplete genome data. Take dinoflagellates as an example of micro-algae for discussion purposes, they are the major causative agents of harmful algal blooms (HABs). Lack of well described genome information of dinoflagellates has limited progress of proteomic studies on these organisms. Nevertheless, 2-DE combined with de novo protein/peptide sequencing could provide an alternative route for identification and annotating proteins of interest in these organisms. However, preparation of high-quality samples of dinoflagellate cell extracts for 2-DE analysis is difficult due to the presence of high endogenous levels of nucleic acids, polysaccharides, salts, pigments, and other interfering compounds. Thus, an optimized protocol of sample preparation is a pre-requisite for obtaining satisfactory and reproducible 2-DE profiles. In light of these perceived problems, we had reviewed current methods available for preparing dinoflagellate samples for 2-DE analysis with some working alternatives from the authors laboratory. Moreover, de-novo protein/peptide sequencing methods by MALDI-TOF MS assisted by effective derivatization protocols were also described.
Keywords: Dinoflagellates, de novo protein/peptide sequencing, MALDI-TOF MS, sulfonation, 2-DE
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