APPLICATION OF PROTEOMIC TOOLS: THE AUTOLYTIC CHANGES OF PORK MUSCULAR TISSUE

Eight protein substances that undergo changes during autolysis were found using the electrophoretic methods with the following mass spectrometric identification. The revealed protein substances have different origin: structural proteins (fragments of troponins T and myosin light chains), and metabolic proteins (creatine kinase, pyruvate kinase and alpha-enolase). The decomposition of the fractions of fast skeletal muscle troponin T in 28.0 kDa, 27 kDa and 26.5 kDa was most pronounced. Identification of constitutive proteins and detection of the products of post — mortem degradation of protein molecules make them suitable candidates for meat quality markers and the following study of these specific fragments will lead to better understanding of the proteolytic activities that take part in the post mortem muscle transformation into meat. УДК /UDC: 637.5’64.05:543.645.6 DOI 10.21323/2414–438X-2018–3–4–32–37 ДЛЯ ЦИТИРОВАНИЯ: Чернуха И.М., Ахремко А.Г. Применение инструментов протеомики на примере изучения автолитических изменений мышеч‐ ной ткани свинины. Теория и практика переработки мяса. 2018; 3(4): 32–37. DOI 10.21323/2414–438X‐2018–3–4–32–37 FOR CITATION: Chernukha I.M., Akhremko A.G. Application of proteomic tools: the autolytic changes of pork muscular tissue. Theory and practice of meat processing. 2018; 3(4): 32–37. (In Russ.). DOI 10.21323/2414–438X‐2018–3–4–32–37 Чернуха И.М., Ахремко А.Г.* Федеральный научный центр пищевых систем им. В.М. Горбатова РАН, Москва, Россия Irina M. Chernukha, Anastasiya G. Akhremko* V.M. Gorbatov Federal Research Center for Food Systems of Russian Academy of Sciences, Moscow, Russia


Introduction
Many factors affect meat quality including keeping animals, their diet, handling and the processes of slaughter, carcass cutting and processing.Until recently, little attention was paid to the molecular or biological aspects of meat quality.Nowadays, the methods of genomics, proteomics and other «omic» approaches open multiple new possibilities.In agriculture, proteomics is becoming more and more important for scientific research.The main tool of proteomics is two-dimensional electrophoresis (2DE).2DE is an electrophoretic method, which allows performing parallel separation and analysis of 500-2000 individual protein molecules, extracted from complex samples.In 2DE analysis the «spotty' structures are formed, where each individual spot represents an individual protein, which migrates to its own specific coordinates according to its specific molecular weight and charge.The spot intensity shows how much of this actual protein was produced by a cell.
During last two decades, 2DE maps of many species and tissues were published [1,2], including maps of pigs [3,4], cattle [5] and chicken [6].Several of these maps were used for a search of molecular markers for meat quality; today, these works remain topical in the food industry.
The aim of this work was substantiation of prospectivity of the proteomics use and discussion of its application in the meat industry by the example of the study of the autolytic transformations in the pork muscles.

Materials and methods
The object of the research was pork M. longissimus dorsi, obtained from the meat processing plant in the city of Rostov-on-Don.The samples were stored at a temperature of 2 ± 2 °C in the conditions of a commercial refrigerator for 120 hours.The samples for analysis were taken after 24, 72 and 120 hours.
Analysis of the protein fractional composition of the samples under investigation was carried out by one-dimensional electrophoresis in 12.5 % polyacrylamide gel in the presence of sodium dodecyl sulfate in a chamber «VE-10» (Helicon, USA).As a standard for electrophoresis, we used the marker of the «Thermo» company (USA), which is a mixture of 11 recombinant proteins (250, 150, 100, 70, 50, 40, 30, 20, 15, 10 and 5 kDa).Staining was carried out using Coomassie G-250.
As the main proteomic technology, two-dimensional electrophoresis (2DE) by O'Farrell with isoelectric focusing in the ampholine gradient (IEF-PAGE) was used; protein detection was carried out by staining with Coomassie R-250.To perform the computer densitometry, two-dimensional electropherograms being in a wet state were used.Their complete digital images and/or images of individual fragments were obtained using a Bio-5000 Plus scanner (Serva, Germany).
Identification of the protein fractions was carried out after trypsinolysis by mass spectrometry MALDI-TOF MS and MS/MS using Ultraflex MALDI TOF mass spectrometer (Bruker, Germany) with the UV laser (336 nm) in the mode of positive ions in the mass range of 500-8000 Da with their calibration by the known peaks of trypsin autolysis.Analysis of the obtained mass spectra of the tryptic peptides was performed using the Mascot program, Peptide Fingerprint option (Matrix Science, USA), with the accuracy of МН+ mass detection equal to 0.01 %, searching in the databases of the National Center for Biotechnology Information (NCBI, USA) [7].

Results and discussion
Proteomics traces back to one-dimensional (1D) electrophoresis proposed by Ulrich K. Laemmli in 1970 [8].At present, this method is used to assess the protein molecular weight distribution in the studied sample.At the early stage of investigations, the obtained results help predict quantitative and qualitative distribution of structural and tissue specific protein molecules and assess an impact of different processes, for example, autolysis (Figure 1).
On the 1D electropherograms, the zones, in which a change in the color intensity of the protein bands can be used to predict their quantity, are marked with the yellow color.However, a serious disadvantage of 1D electrophoresis is the fact that analysis is limited only to the approximate detection of the molecular weight and one protein band can contain several protein molecules with the same molecular weight.
Patrick O'Farrell managed to increase the effectiveness and resolving power.In 1975, he proposed the 2DE method [9], in which proteins are separated by two different physico-chemical parameters [10,11].
As more than 20 % of muscle mass (or meat) is composed of proteins, the presence of interrelation between muscle proteins and meat quality is evident.It was found that proteins (Figure 2) such as pyruvate kinase (1), alphaenolase (2), creatine kinase (3), troponins T (4,5,6) and myosin light chains (7,8) undergo changes upon the autolytic transformations of pork muscles.
Proteins identified by mass spectrometry are presented in table 1.The revealed spots have different origin: structural proteins, namely, troponins T and myosin light chains, as well as metabolic proteins such as creatine kinase, pyruvate kinase and alpha-enolase.Three spots were identified as the fragments of troponin T in 28.0 kDa, 27 kDa and 26.5 kDa, and, as shown in Fig. 2, they decreased in intensity, while full-size troponin T (43 kDa) was not found.Extensive degradation of troponin T is well-known and it is believed that it is linked with meat tenderness [12].
A change in the spot intensity of myosin light chains (MLCs) presumably affects the meat color and its tenderness as MLCs regulate activation of the myofilament by phosphorylation by calcium ion binding.
The intensity of the spot that corresponded to creatine kinase increased by the 3rd day, and decreased again by the 5th day.This is associated with energy transduction in tissues with high variable energy requirements such as skeletal muscle.
In addition, an increase in the large allosteric fragment of pyruvate kinase, which regulates glycolysis by linking the substrate, phosphoenolpyruvate, and one or several active molecules was observed.An increase in the concentration of pyruvate kinase suggests a change in its activity as a result of non-covalent attachment of a specific metabolite (amino acid, nucleotide and others) to its allosteric center.
A significant increase in the intensity of alpha-enolase as a participant of biosynthesis of amino acids can afterwards provoke an increase in their quantity in the muscle tissue in the process of autolytic changes.
Therefore, this study provides a better understanding of proteome changes during postmortem transformations in porcine muscles.The structural proteins and enzymes linked with the glycolytic pathway and energy metabolism were modified.The further research of post-translational modifications of related proteins and changes in the level of metabolites is needed.

Conclusions
Two-dimensional electrophoresis is an effective method for meat protein identification.Eight protein substances that undergo changes during most mortem autolytic transformations were studied by the proteomic methods.It was shown that the main sign of the autolytic processes was an appearance of the fragments of fast skeletal muscle troponin T due to a continuous decrease in the fragments of peptides of С-and N-terminal parts of molecules.
It was found that metabolic enzymes degrade during autolysis.The role of these enzymes in the meat post-mortem metabolism has not been completely elucidated, and the results of the performed study can help focus on these aspects of post-mortem changes and reveal their relationship with meat quality.

Table 1 . 2 *
Results of the mass-spectrometric identification (MALDI-TOF MS and MS / MS) of protein fractions showing changes in 2DE of proteins from M. longissimus dorsi of Sus scrofa during autolysis (numbers of proteins correspond to Figure Мм -molecular weight, pI -isoelectric point ** Mm/pI (exp.)-scores obtained by the results of the electrophoretic mobility on DE, Mm/pI (calc.)-estimates made on the basis of the data on the amino acid sequence with consideration for signal peptide removal, but without regard for other post-synthetic modifications using the program ExPASy Compute pI/Mw tool

Figure 2 .
Figure 2. 2DE fragments with revealed signs of the autolytic changes in the studied pork samples