Dried seafood products and traditional Chinese tonic foods are commonly found in Chinese markets. They are key ingredients in Chinese cooking and some are highly valued due to their nutritional and medicinal properties. The market price of the caterpillar fungus species Ophiocordyceps sinensis
is approximately HK$
50/g in China [1
], for example. Moreover, the general selling price of dried abalone (sometimes sold in slices) ranges from a few thousand to over 10,000 Hong Kong dollars per catty (604.79 g) [2
]. Therefore, highly valued genuine species are often adulterated with low-valued species that are considerably cheaper. The Hong Kong Customs and Excise Department, in 2010, discovered more than 100 catties of fake dried abalone slices in a series of raids on 31 dried seafood retail shops in Hong Kong [2
], for instance. The fake dried abalone slices were all found to be dried whelk slices instead. Customers would have difficulty identifying the adulterants based on the physical appearance, colour, taste, and texture of the food products alone without experimental examination. Therefore, the development of testing technology is crucial for the authentication of these valuable dried food products.
Proteomic technologies are powerful tools for the large-scale study of the proteins encoded by an organism [3
]. These technologies provide a platform to determine biological mechanisms and discover protein biomarkers [4
]. Gel-free approaches, such as multidimensional liquid chromatography and protein arrays, have been developed in recent years and have further enhanced the research tools available in proteomic analysis [6
]. However, two-dimensional gel electrophoresis (2-DE) remains one of the most widely used protein separation techniques due to its high resolution and low cost [8
]. Two-dimensional gel electrophoresis is a powerful technique that separates a mixture of proteins into individual protein spots according to their isoelectric point (pI) and molecular weight (MW) under defined conditions. The protein spots can be easy visualised on the gel, and they can be isolated and identified using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) with a peptide mass fingerprint (PMF) and de novo peptide sequencing approaches. Proteomics analysis has been widely used in food authentication studies and food technology research [9
], and 2-DE has been used to analyse the proteome of traditional Chinese tonic foods such as ganoderma and caterpillar fungus [11
]. Although the production of 2-DE gel includes many steps, obtaining a quality protein sample remains the most important precondition for high-quality 2-DE. Samples including compounds such as nucleic acids, polysaccharides, lipids, pigments, and salts can severely affect the isoelectric focusing (IEF) process (the first step of 2-DE), which leads to smearing and streaking in the 2-DE gel [13
]. Thus, tedious and lengthy preparatory steps are required to remove these endogenous contaminants.
The clean-up procedures are typically time consuming and laborious, possibly taking several days. The trichloroacetic acid (TCA)-acetone method is a conventional approach used to extract protein from samples with complex matrices, especially plant samples [17
]. It is effective in the removal of interfering compounds, and it produces high-quality protein samples due to its effective precipitation of proteins. Although TRIzol reagent (a phenol-guanidine isothiocyanate solution) is typically employed to isolate RNA from cells and tissues, it has been widely applied to extract proteins for 2-DE. This TRIzol-based extraction method has gained prominence and attracted attention due to its promising performance in removing contaminants from protein samples and, thus, facilitating high-quality 2-DE. The TRIzol-based extraction method was first applied successfully on halophilic protein samples for the generation of 2-DE in 2006 [19
]. This protein extraction method has subsequently been widely used in 2-DE for a wide range of samples, including rat spinal cord [20
], human/animal cell lines [21
], dinoflagellates [11
], mites [29
], plant tissue [30
], clinical samples [31
], marine animals [34
], and reef corals [37
] (Table 1
). This TRIzol-based method has gained popularity for 2-DE because it offers several advantages [19
]. Compared with other conventional methods, the TRIzol-based method is convenient and fast. Soluble protein samples can usually be obtained in less than 4 h. Additionally, it effectively removes interfering compounds and renders samples fully compatible with IEF. More importantly, DNA and RNA can be extracted together with the proteins from a single sample. Some studies have demonstrated that analysing all such macromolecules from a single specimen would enable the integration of transcriptomic and proteomic data and thus provide a more comprehensive view of the regulatory mechanisms [20
]. However, few studies have investigated protein extraction from dried seafood and traditional Chinese tonic foods for 2-DE.
The authors aim to evaluate the effectiveness of the TRIzol-based protein extraction method for 2-DE for dried seafood and traditional Chinese tonic foods. Four valuable dried food products—two dried seafood products (abalone slices and whelk slices) and two traditional Chinese tonic foods (ganoderma and caterpillar fungus)—are used in the study. Gels produced though the TRIzol-based method are compared with those produced through the TCA-acetone method in terms of protein yield, spot number, spot intensity, and resolution. Moreover, TRIzol-based 2-DE profiles of abalone slices and whelk slices are compared and corresponding differentially expressed proteins are identified. The results are intended to provide a useful foundation for the development of new authentication methods for dried seafood and traditional Chinese tonic foods through gel-based proteomic analysis.
The generation of high-quality two-dimensional gel electrophoresis (2-DE) profiles is important for gel-based proteomic analysis. The authors evaluated the use of TRIzol protein extraction to produce 2-DE from two dried seafood products and two traditional Chinese tonic foods. Although lower protein yields were observed in the TRIzol extraction of abalone and whelk samples, high-quality 2-DE gels were obtained for all four samples through the TRIzol method, and the quality was comparable to that of the 2-DE gels generated through TCA-acetone precipitation in terms of numbers of spots, background signal, and resolution. TRIzol-prepared gels showed clearer backgrounds, higher intensity, and more spots than did the gels prepared using TCA-acetone in specific areas. The feasibility of identifying differentially expressed proteins from the 2-DE comparison of abalone and whelk samples was also demonstrated. This study established that TRIzol-based protein extraction provides simple, fast, reliable, and feasible sample preparation using a 2-DE–MS workflow for the proteomic analysis of dried seafood products and traditional Chinese tonic foods.