Meat and meat products will always play an important role in the human diet as a source of high-grade protein. There is a wide variety of meat processing products, the range of which is regularly updated. Depending on the processing technology, meat proteins undergo extensive modifications that affect their quality, shelf life, nutritional properties, and health effects. At the same time, tenderness and juiciness are the criteria most judged by consumers (Cao et al., 2020; Hollung et al., 2007).

These criteria are influenced by the following factors: genetics, environment, animal welfare, and further processing. The molecular mechanisms underlying such processes are still of interest. However, genetic information remains static throughout the life of the body, while the protein composition is dynamic and changes depending on factors affecting protein synthesis or degradation (Peng and Gygi, 2001). Thus, proteomics analysis makes it possible to better understand the molecular mechanisms that occur in tissues and to predict the most important characteristics, particularly in the case of meat (Bendixen, 2005; Bendixen et al., 2005; Zamaratskaia and Li, 2017).

Proteomic methods over the past decade have found increasing application in various fields of science and agriculture. To date, proteomic tools have improved significantly, and mass spectrometric methods are being developed very rapidly. Most proteomic methods are based on the separation of proteins in at least two directions, using chromatographic or electrophoretic methods, with further identification using mass spectrometry methods (Soares et al., 2012; Suman et al., 2014). In this paper, attention is focused on the method of two-dimensional gel electrophoresis (2-DE), because it is still an excellent way to visualize protein components and widely used in meat science, with an emphasis on sample preparation and aspects of isoelectric focusing (IEF).

Traditionally, 2-DE is carried out by fractionation of proteins according to two different physicochemical parameters. In the first direction, proteins are separated by charge according to their isoelectric point with IEF, and then in the second direction by their molecular weight with polyacrylamide gel electrophoresis (PAAG). Some variations of conducting 2-DE are discussed below to identify the most optimal variant for analysis of the meat proteome.

In most scientific works, IEF is performed using a thin layer of gel deposited on a plastic substrate (IPG Dry Strip) to speed up and simplify the process. (Naveena et al., 2017; Di Luca et al., 2016; Lee, Saraygord-Afshari and Low, 2020). However, the glass-tube IEF, despite process complexity, has an advantage in the resolution and protein loading volume in the gel (Matsumoto et al., 2019). Based on foregoing, for this experiment, we selected glass-tube IEF.

In accordance with the experimental design, depicted in Figure 1, four 2-DE variations were performed; the obtained electropherograms are presented in Figure 2.

A different distribution of pig muscle protein was noted for molecular weights (MW) and isoelectric points (pI).

In the classical version of IEF (O'Farrell, 1975; Hirano, 1982; Kimura et al., 2003), when 0.02 M NaOH is in the upper chamber of the electrophoretic cell (Figure 2A), good protein separation is observed within a pI range of 5 to 6.5, such as tropomyosin beta chain (Mw 33.5 kDa, pI 4.80) (D´Alessandro et al., 2011; Peng et al., 2013), tropomyosin alpha-3 chain (Mw 33.5 kDa, pI 4.71) (Davoli et al., 2000), myosin light chain 3-like (Mw 22.0 kDa, pI 5.24) and myosin light chain 1/31 (Mw 21.0 kDa, pI 5.80) (Montowska and Pospiech, 2012; Kovaleva et al., 2013).

Moreover, on two-dimensional electropherograms with incubation in buffer A (Figure 2B), these proteins are most clearly identified (Ros et al., 2002; Montowska and Pospiech, 2007; Paredi, Mori and Mozzarelli, 2018). At the same time, in the alkaline zone of the gel, a blurred image of protein fractions is observed. A completely different gel was obtained when the current direction changed during the IEF, when 0.01 M orthophosphoric acid was in the upper chamber of the electrophoretic cell. Clear and well-defined protein spots were obtained with uniform distribution over the entire gel area (Chernukha et al., 2017). Presumably, this was because proteins with acidic and neutral pI are more highly represented in muscle tissue, resulting in a higher proportion of positively charged proteins. Since the current always goes in the direction from “plus” to “minus”, when changing the direction of the current flow, it is easier for positively charged proteins, which are always layered on top to move to the lower chamber of the electrophoretic cell, where a negative charge (cathode) is created (Colangeli et al., 2018).

Interestingly, during incubation in equilibration buffer I and II (Figure 2C), protein spots with a molecular weight greater than 50 kDa were better visualized (Vasilevskaya and Akhremko, 2019). In contrast, when incubated in buffer A (Figure 2D), protein spots with a molecular mass of less than 40 kDa were better visualized. The latter variation was used in a study by Kovalev (Kovalyov et al., 2006; Zvereva et al., 2015), and analysis of the results found that this method, as in the case of incubation in equilibration buffers, allows the best detection of muscle tissue proteins.

Major structural proteins of pig muscle tissue (Figure 3) were found (Montowska and Pospiech, 2012) and subjected to densitometric analysis (Table 1). The Fold change index (Persike et al., 2018) was calculated. Fold change index is the ratio between the volume of protein spots with the highest average value and the lowest average value. Eight fractions were detected (≥2-fold change, p <0.05) with increased spot volume by at least 2 times compared with the classical version of the IEF (marked * in Table 1).

Thus, the variation of the IEF, when sodium hydroxide is in the upper chamber of the electrophoresis cell, has a lower resolution and does not properly detect protein fractions in the alkaline zone. So, in Figures 2A and 2B, proteins in the right half of the gel are noticeably out of focus. It is necessary to increase the number of volt-hours by at least 40% (up to 7 – 8 hours) for better protein separation in this area.

After changing the direction of electric current during the IEF, the process takes 4 hours. As a result, at least two times more protein spots are detected on 2-DE, including major structural muscle proteins, such as glyceraldehyde- 3-phosphate dehydrogenase (Han et al., 2019), troponins group (Mora et al., 2016; Drousiotis et al., 2020), 3-hydroxyacyl-Coenzyme A dehydrogenase, beta-enolase and others (Nolan et al., 2019).

The results of electrophoretic studies showed that the best option for the separation of meat proteins is to change the direction of the current. Thus, when the anode buffer (0.01 M orthophosphoric acid) is in the upper chamber of the electrophoretic cell, and the cathode buffer (0.02 M sodium hydroxide) is in the lower one, the most informative picture is obtained. Incubation in both buffer A and equilibration buffers can also be used.

Densitometric analysis showed that the use of new parameters allows us to identify a larger number of proteins (almost 2 times). An increase in the color intensity of certain fractions is also noted. Thus, the proposed variation of the IEF can be used as the main one for muscle proteins electrophoretic analysis, since it requires less time and has a higher resolution.