Abstract
Two-dimensional gel electrophoresis (2-DE) is a major element of conventional gel-based proteomics, which resolves complex protein mixtures. Protein extraction with the removal of interfering substances from the sample remains the key to producing high-quality 2-DE profiles. Marine dinoflagellates contain large endogenous amounts of salts, nucleic acids, polysaccharides, phenolic compounds, pigments, and other interfering compounds. These substances are detrimental to the quality of gel images. Protein preparation using TRIzol reagent is a promising method for producing high-quality 2-DE profiles for dinoflagellate samples. In addition to its remarkable performance, the TRIzol method’s several advantages have made it a popular and widely used method in the field of 2-DE sample preparation. Nonetheless, the quality of 2-DE of samples from certain dinoflagellate species is not as high as previously reported when the same TRIzol protocol is applied. Therefore, modifications to the original TRIzol method are required to remove interfering substances from those challenging dinoflagellate samples. In this study, the original TRIzol method and four modified methods, namely the aliquot TRIzol method, re-TRIzol method, TRIzol method with a commercial clean-up kit, and TRIzol method with trichloroacetic acid/acetone precipitation, were compared. Performance of these five methods in terms of protein yield, background signal, and resolution and number of protein spots was investigated on samples from two benthic Prorocentrum species: P. lima and P. hoffmannianum. Our results demonstrated that high-quality 2-DE could be achieved from P. lima samples prepared using both the original TRIzol method and the TRIzol method with a commercial clean-up kit. However, the original TRIzol method failed to produce high-quality 2-DE profiles for P. hoffmannianum samples. Among the four modified TRIzol methods, only the TRIzol method with a commercial clean-up kit could yield substantially improved high-quality 2-DE profiles for P. hoffmannianum samples. This combination of the conventional TRIzol method with a commercial clean-up kit potentially represents a promising protein extraction methodology for obtaining high-quality 2-DE profiles for difficult dinoflagellate samples.
Highlights
Harmful algal blooms (HABs) are natural phenomena that occur as a result of the massive growth of microalgae in water bodies
Modifications of the original TRIzol method are required to improve the quality of proteins extracted from these difficult dinoflagellate samples
We proposed four modification methods, namely the aliquot TRIzol method, re-TRIzol method, TRIzol method with a commercial clean-up kit, and TRIzol method with trichloroacetic acid (TCA)/acetone precipitation (Figure 1)
Summary
Harmful algal blooms (HABs) are natural phenomena that occur as a result of the massive growth of microalgae in water bodies. Eng. 2020, 8, 363 degradation of proteins and enhances the recovery of the samples without the need of additional protease inhibitor [7,9] Owing to these advantages, protein extraction through the use of TRIzol reagent for 2-DE has become popular and has been applied in several proteomic studies of various algal species (Table 1), such as planktonic P. donghaiense [10], the toxin-producing A. catenella (responsible for paralytic shellfish poisoning) [11,12,13,14], yessotoxin-producing Linulodinium polyedrum [15], diarrhetic shellfish poisoning toxin producing Prorocentrum [16], unarmed dinoflagellate, Gymnodinium [17], and Eustigmatophyte, Microchloropsis gaditana [18]. Proteins of the benthic dinoflagellate species P. hoffmannianum prepared using the TRIzol method produced gel images of unsatisfactory quality [16]. Species that our team demonstrated as workable to produce high-quality 2-DE profiles using TRIzol method (data not shown)
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