Proteomics Analysis and Identication of Critical Proteins and Network Interactions That Regulate the Specic Deposition of IMF of Jingyuan Chicken

Background:Improving the production eciency and quality of broiler chickens has become a hot topic in molecular breeding of the broiler industry. There are several factors, among which intramuscular fat (IMF) content is one of the essential factors determining chicken quality. The chicken carcass weight of 40-50% consisted of the leg muscles and breast muscles, and the research on chicken meat quality was performed on that part. Jingyuan chicken is one of the high-quality local chicken species in China. However, the molecular mechanism of IMF specic deposition in different parts of Jingyuan chicken is still unclear. In order to screen and identify the proteins that regulate IMF specic deposition in different parts of Jingyuan chicken and analyze network interaction, the present study selected 180-day-old Jingyuan chicken as the research material, using proteomic techniques and PRM experimental methods to carry out basic research on leg muscle and breast muscle. Results:Three key signaling pathways and ten essential functional proteins regulating IMF specic deposition of Jingyuan chicken were identied, while the molecular mechanism of the deposition of proteins was explained, and analyzed the network interaction between these key functional proteins. Conclusions:The results of this study are of great scientic value and theoretical signicance not only for improving chicken quality but also for molecular breeding of high-quality local chickens.


Background
Poultry meat has become the second-largest meat consumption in China, and its demand is increasing year by year, which shows that poultry meat has become an essential item to human beings (Dengfei et al. 2016). The growth of poultry production has been outstanding due to the remarkable achievements made by researchers through genetic improvement techniques on the growth rate, feed remuneration, and muscle yield of poultry (Laura et al. 2017;Zhichao et al. 2018). Unfortunately, it is regrettable that the quality of poultry meat has continued to decline as a result of the development in poultry production and the constant growth in muscle production (Muazzez et al. 2016). With the continuous improvement of the human dietary structure, the demand for meat products has gradually changed from quantity to quality, in particular the quality and avor of poultry meat. Therefore, improving both the quality and quantity of broilers has become a hot topic in the current research. Chicken yield and meat quality traits are closely related to living body weight, carcass weight and other characteristics, of which between 40 and 50 percent of carcass weight is composed of leg and breast muscles (Chen Gangyao et al. 2012), Therefore, to improve the quality of chicken one should concentrate on breast and leg muscles of chicken. It has been found that there are signi cant differences in meat quality between leg muscle and breast muscle.
Furthermore, several other parameters, including pH, water capacity, cooking loss, muscle ber, intramuscular fat (IMF) content, and inosinic acid, are responsible for determining the quality of meat .
IMF refers to the fat distributed among muscle bers in muscle tissue. The content of IMF plays a key role in the avor, freshness, juiciness, taste, tenderness, and color of meat (MA et al. 2015). Studies have shown that IMF is derived from adipocytes between the myofascial membrane and myointimal, and these fat cells are derived from myogenic PDGFRα+ progenitor cells (Guan et al. 2017;  and MyoD-directed differentiation of Myf5-and Pax3-non-myogenic progenitor cells (Liu et al. 2012).
Previous studies have suggested that the IMF content can be determined by both chronologically (Lin Yaqiu et al. 2015), and heritability (H2 = 0.5) ). The studies on the molecular mechanism of chicken IMF speci c deposition are exhibited into four aspects which are as follows: (i) The biochemical mechanism of IMF affecting meat avor was analyzed by previous researchers ).
(ii) Considering the dimension of development, we have explored the deposition rules of IMF in vivo and the primary functional genes and signal pathways regulated at various stages. For example, it was found that the late embryonic stage is also one of the critical periods for intramuscular fat deposition. Studies have identi ed the essential roles of PPAR, fatty acid oxidation, cell connectivity, and other related pathways in intramuscular fat deposition (Zhang et al. 2017).
(iii) The fat deposition on different parts of the body roughly elaborated the molecular regulatory mechanism for differential fat deposition in breasts, leg muscle, and abdominal fat was compared with each other (Cui et al. 2018).
(iv) It is con rmed that genomic breeding methods have some potential in improving intramuscular fat But in general, the regulation of IMF is the product of the interaction of multiple signaling molecules. The current research on IMF deposition mostly remains at the genetic level and the difference in content between varieties. However, the regulation of IMF speci c deposition for critical proteins in different parts are not available as few related studies on molecular mechanisms and interactions between proteins are obtained so far.
China has abundant high-quality local broiler germplasm resources, with delicious meat, high tolerance of disease, and strong adaptability. Jingyuan chicken is one of the ve district-level livestock and poultry genetic resources in the Ningxia Autonomous Region, and it is also an outstanding broiler breed in the Animal and Poultry Genetic Resources from China. After doing a lot of basic research on the production performance and meat quality of Jingyuan chicken, we found that the IMF content of Jingyuan chicken was higher than that of other breeds and signi cantly greater than that of fast large-scale broiler AA chicken. This biological characteristic is consistent with the results of a comparative study on the intramuscular fat deposition of other local broilers and AA Broilers by many researchers. Besides, Jingyuan chicken also contains certain unsaturated fatty acids (eicosapentaenoic acid, docosapentaenoic acid, 32-carbohexaenoic acid, etc.) that have exceptional nutritional value for the human body. However, the mechanism of the speci c deposition of IMF in different parts of Jingyuan chicken is not clear. The particular expression and regulation of proteins in its body and the network of interactions need to be deeply analyzed.
In summary, to improve chicken quality and production performance, in particular, increasing the IMF content in chicken, has become a hot topic in current research. IMF deposition is in uenced not only by spatiotemporal but also by heritability. The occurrence, development, and deposition of IMF cells are extremely complex, including an interactive physiological and biochemical process. It is di cult to pass a gene, protein expression pattern, or a signal transduction pathway to clarify the mechanism of IMF speci c deposition. Nonetheless, the effect and control of genetic, environmental, and nutritional factors on animal intramuscular fat deposition are inseparable from the involvement of main functional proteins and thereby associated signaling networks in the animal body. Although the animal molecular nutrition pathway can also regulate lipid metabolism and intramuscular fat deposition, it is ultimately the result of speci c protein expression and translation modi cation . Therefore, the use of proteomics technology to screen and identify essential proteins and signal network pathways that control IMF speci c deposition in different parts of the chicken, providing relevant molecular data for further improvement of chicken quality and accurate broiler molecular breeding.
Based on the previous research of the research group, we selected the high-quality local broiler breed-Jingyuan chicken as the research object. We used the label-free proteomics technique and parallel reaction monitoring method (PRM) to screen and identify the critical functional proteins that regulate the speci c deposition IMF the leg muscle and breast muscle of Jingyuan chicken. The results have not only signi cant biological and economic value to improve the avor and quality of chicken but also have typical representative signi cance to the molecular breeding research of high-quality local broilers.

Animals and samples
In this research, 30 (hens) of 180-day old white feather Jingyuan chicken with an average live weight of 2.5 ± 0.23 kg were selected from the breeding center of Chaona chicken in Pengyang County of Ningxia Hui Autonomous Region. Their muscles of the leg and breasts were collected in an aseptic environment and stored at -80 ℃ for future use.
In accordance with the "Environmental Protection Law of the People's Republic of China" and the "Animal Epidemic Prevention Law of the People's Republic of China", we burn and bury animal carcasses after experiments to prevent them from causing environmental pollution and harm to human health.

Protein extraction and enzymatic hydrolysis
The sample was removed from the -80°C refrigerator and ground it thoroughly in a mortar with liquid nitrogen. After grinding into powder, four times volume lysis buffer (8M urea, 1% protease inhibitor) was added for ultrasonic lysis. Then centrifuged of 12000 g at 4°C for 10 min, cell debris was removed, and the protein concentration was determined by the BCA kit. Upon dilution of the lysed proteins with dithiothreitol and iodoacetamide, trypsin was added at a mass ratio of 1:50 (trypsin: protein), and enzymatic hydrolysis was performed overnight at 37℃. Then trypsin was then added at 1:100 mass ratio (trypsin: protein) to continue enzymatic hydrolysis for 4 h.

Liquid chromatography-mass spectrometry analysis
The peptide was dissolved in an aqueous solution containing 0.1% formic acid and acetonitrile solution containing 0.1% formic acid by liquid chromatography mobile phase A and mobile phase B, respectively.
Then peptide was separated by Nano ultra-high performance liquid system and injected into capillary ion source for ionization and then analyzed by time to pro-mass spectrometry.

Database search and bioinformatics analysis
First, MaxQuant (v1.6.6.0) was used to retrieve the mass spectrometry data, and Gallus_gallus_UniPort (29475 proteins) database was used. Some parameters in the database were modi ed and set, including the enzyme restriction method Trypsin/P, to effectively eliminate the in uence of contaminated proteins on the identi cation results and reduce the false positive rate (FDR). The number of missing bits was set to 2, while Firstsearch obtained the rst order parent ion mass error tolerance, and the Main search was set as 70 ppm and 70 ppm, respectively. The mass error tolerance of secondary fragment ions was set as 0.04da. The xed modi cation was set for cysteine alkylation, and variable modi cation was established for methionine oxidation and N-terminal acetylation and deamidation of protein. The FDR for protein identi cation and PSM identi cation was set at 1%. The potential connections and differences on their speci c functions (GO, KEGG pathways, protein domains, etc.) were analyzed for biological information based on the functional enrichment and clustering speci city of different groups with differentially expressed proteins (or differentially expressed proteins with different differential multiples),

Parallel reaction monitoring (PRM)
Speci c proteins related to breasts and leg muscles were quanti ed in LC-PRM/MS by PRM. The results obtained from PRM were calibrated using Xcalibur™ software. The peptides were separated by an ULTRA high-performance liquid system, injected into an NSI ion source for ionization, and then analyzed by Q ExactiveTM Plus mass spectrometry.

Results
Mass spectrometry data analysis of proteome in Jingyuan chicken Leg muscle and Breast muscle Label-free quantitative proteomics was used to analyze the Jingyuan chicken leg muscle and Breast muscle tissues. 265,391.0 secondary protein spectrograms were identi ed by LC-MS/MS mass spectrometry, and 85811.0 effective spectrograms were obtained after data search, with the utilization rate of 32.3%. After spectrum analysis, 15,139.0 peptides were identi ed, among which 12,819.0 speci c peptides were identi ed. After examination and comparison, 1940.0 proteins were identi ed, among which 1317.0 proteins could be quanti ed ( Figure 1A). The results showed that the sequence length of detected peptides was mainly distributed between 8 and 32 ( Figure 1B). The error between the actual and theoretical values of the identi ed protein-peptide relative molecular weight was concentrated around 0, less than 20 ppm ( Figure 1C). The analysis found that most peptides were less than 200 kDa, and these proteins covered 80% of the identi ed protein sequences ( Figure 1D). The above data indicated that protein identi cation results were relatively ideal, and the subsequent data analysis could be continued.
Screening and principal component analysis of differential proteins in leg and Breast muscles of Jingyuan chicken By using mass spectrometry, in each protein, the signal abundance was analyzed, and the LFQ intensity value of that protein was obtained using a non-standard quantitative calculation method. Then the calculation of the relative quantitative value of each sample was done according to the protein LFQ intensity value between different samples. The calculation principle of differential protein expression is to screen for differential proteins with a p-value of < 0.05. When the differential protein expression changes by more than 2, it indicates that the differential protein is upregulated during life-long activities. When the differential protein expression varies less than 1/2, it shows that protein is downregulated during life activities. According to the principle of protein differential expression screening, a total of 190 differentially expressed proteins were found in the muscles of the leg and breasts. Compared to the breast muscles, 121 proteins in the leg muscles were upregulated, and 69 types of proteins were downregulated (Figure 2A, B ). The principal component analysis was carried out on 190 differential proteins identi ed in Jingyuan chicken leg muscle and breast muscle, as shown in Figure 2C. The variance contribution rate of the rst principal component PC1 was 53.8%, the variance contribution rate of the second principal component PC2 was 17.4%. The contribution rate of PC1 and PC2 reached 71.2%, re ecting most of the information on the change of differential proteins, and the proteome of the two parts was signi cantly different.
Functional classi cation and GO enrichment analysis of differentially expressed proteins in Jingyuan chicken leg muscle and breast muscle To further study the biological functions performed by differentially expressed proteins in Jingyuan chicken leg and breast muscles, we performed Gene Ontology (GO) function annotation and enrichment analysis of 190 differential proteins. Mainly through the biological process (BP), cellular component (CC), and molecular function (MF) are three aspects of the differentially expressed proteins that have been elaborated. Figure 3 shows that differential proteins are mainly involved in cellular processes (116 proteins, 22%), single-organism process (112 proteins, 21%), metabolic processes (93 proteins, 18%) ), biological regulation (47 proteins, accounting for 9%), cellular component organization or biogenesis (33 proteins, accounting for 6%) and other biological processes. In CCs, differential proteins are mainly involved in the cell (114 differential proteins, accounting for 30%), organelle (87 proteins, accounting for 23%), macromolecular complex (51 proteins, accounting for 14%), membrane (49 proteins, accounting for 13%), extractor region (38 proteins, accounting for 10%) and other cell components. In MFs, differential proteins mainly participate in binding (123 proteins, accounting for 45%), catalytical activity (104 proteins, accounting for 38%), transportation activity (16 proteins, accounting for 6%), structural molecular activity (11 proteins, accounting for 4%) and other molecular functions.
Further, the subcellular localization of 190 differentially expressed proteins found in leg muscle and breast muscle of Jingyuan chicken. The differential proteins were mainly located in the cytoplasm (92 proteins, 48.42%), mitochondria (28 proteins, 14.74%), nucleus (24 proteins, 12.63%), extracellular (21 protein, 11.05%), cytoplasm nucleus (12 proteins, 6.32%), plasma membrane (9 proteins, 4.74%), other (4 proteins, 2.11%) organelles, as shown in Fig. 4. For systematically analyzing the differential protein functions identi ed in Jingyuan chicken leg and breast muscles, COG (Cluster of Orthologous Groups of proteins) was used to perform gene function annotation and genome evolution analysis on 190 differential proteins. Figure 5 shows the results obtained from the study. The Histogram shows participation in energy production and conversion (15 proteins), carbohydrate transportation and metabolism (15 proteins), lipid transportation and metabolism (13 proteins), cytoskeleton (12 proteins), general function prediction (9 proteins), translation, ribosomal structure and biogenesis (5 proteins), intracellular tra cking, secretion and vesicular transport (5 proteins), etc. The biological processes with the highest frequency of proteins are primarily energy production and conversion (15 proteins), carbohydrate transport, and metabolism (15 proteins), lipid transport, and metabolism (13 proteins). The biological processes with the lowest frequency of occurrence mainly include post-translational modi cation, protein turnover, chaperones, coenzyme transport and metabolism, Inorganic ion transport and metabolism, secondary metabolites biosynthesis, transport, and catalyst, etc. However, in the annotation of gene function and genome evolution analysis, 90 different protein functions have not been determined.
KEGG pathway analysis of differentially expressed protein in Jingyuan chicken leg muscle and breast muscle Research shows that many types of proteins in the body perform their biological functions through interaction, and a single protein cannot independently perform its biological functions as the interaction of these proteins is connected by signal pathways. Therefore, the analysis of different pathways of proteins can help us to understand the biological functions more systematically and comprehensively. The regulatory mechanism of protein-protein interaction involves a change of character or occurrence of diseases. The KEGG analysis method may well classify proteins, identify the metabolic or signaling pathways in which these proteins may participate, and obtain information about the proteins involved in a series of biological processes from the cell surface to the nucleus, which ultimately helps us understand the entire organism. KEGG pathway enrichment analysis of 190 proteins differentially expressed in Jingyuan chicken leg muscles and breast muscles showed that 190 differential proteins involved a total of 286 signaling pathways, of which these differential proteins involved more than 20 signi cant pathways: gga01100 Metabolic pathways, gga00500 Starch and sucrose metabolism, Figure  6 depicts gga00010 Glycolysis/Gluconeogenesis, gga00071 Fatty acid degradation, gga01212 Fatty acid metabolism, gga01200 Carbon metabolism, gga00280 Valine, leucine and isoleucine degradation, gga03320 PPAR signaling pathway, etc. Thus, proteins with a signi cant difference between Jingyuan chicken leg muscle and breast muscle are mainly enriched by gga000500 starch and sucrose metabolism, gga00071 Fatty acid degradation, gga01212 fatty acid metabolism, gga03320 PPAR signaling pathway, etc.
Parallel reaction monitoring (PRM) validation of the differential expression protein between leg muscle and breast muscle of Jingyuan chicken According to the information on the differential protein peptides in combination with GO analysis and KEGG pathway analysis, 23 target proteins were selected for PRM quanti cation. Due to the characteristics of some proteins and the abundance of their expression, only 21 of them have been quanti ed. It was found that 10 of the 21 quanti ed proteins were related to fat deposition, namely E1BTT4, E1C0Q5 (ACAT1), F1NC38 (ACADL), Q5F420 (ACSL1), A0A1D5P3S9 (ECI1), Q5ZL56 (ACADS), F1NUQ3 (FABP3), A0A1D5PL36 (ACAA2), F1NR44 (ECHS1), Q5ZIR7 (FABP5) and other ten proteins. These proteins were enriched in "gga00071Fattyaciddegradation, gga01212Fattyacidmetabolis, gga03320PPARsignalingpathway" network pathways that regulate fat deposition, as shown in Table1. The activity of related proteins that regulate fat deposition in Jingyuan chicken leg muscles was found to be signi cantly more potent than that of breast muscles, further explaining the critical reason why the IMF content of leg muscles was higher than that of breast muscles.

Network Interaction Analysis of IMF-speci c Deposited Key Proteins in Jingyuan Chicken Leg and breast Muscles
Protein is the primary executor of life activities, and the interaction between proteins plays a vital role in regulating life activities. Both STRING and Cytoscape software were used to analyze the network interactions of IMF speci c deposition proteins that regulated the leg and breast muscles of Jingyuan chicken to obtain a protein-protein interaction network map. Figure 7 depicts that there are eight proteins involved in the protein-protein interaction network, and these eight proteins form 19 network interaction paths, indicating that IMF deposition is a very complex and ordered biological process.

Discussion
Meat quality is a comprehensive economic character, including avor, texture, nutrition, safety, and so on. Many factors are affecting the quality of domestic chicken, including muscle pH, temperature, the water activity of the reaction system, the interaction of avor, and the composition of the meat matrix. The factors of production management mainly include breed, age, sex, dietary factors, feeding environment, feeding system, slaughter process, conditions for carcass storage, cooking methods, etc. which have different degrees of in uence on the taste of poultry meat ( Genetic and nutritional factors regulate IMF deposition, which has a medium genetic factor (h2 = 0.5) in the present studies. Differences in animal species, sex, age, tissue location, etc., cause discrepancies in IMF deposition (Li Huifang and Chen Kuanwei 2004;Jiang Ruirui 2010). Studies have found that the IMF content of leg muscles is signi cantly higher than that of breast muscles, which is 3-4 times that of breast muscles (Li Wenjuan 2008  The high-intensity breeding selection causes the growth speed of the broiler rapidly, almost reaching its physiological limit, leading to a major reduction in the quality of the meat. Rapid growth causes excessive fat accumulation in the abdomen and internal organs, resulting in a certain amount of waste of resources and environmental pollution. Therefore, improving the IMF and the meat quality while broilers are rapidly growing is a hot topic in the current research direction of broiler breeding. Based on the previous results obtained in this project, Jingyuan chicken, a high-quality local chicken species in China, was selected as the objective of the present research. By studying lab free proteomics on the leg and breast muscles of Jingyuan chicken, it was found that there were 190 differentially expressed proteins between them, of which 121 proteins were upregulated, and 69 proteins were downregulated. According to the GO analysis of 190 differential proteins, 93 proteins participated in the biological process of metabolic process, while 47 proteins participated in the biological regulation. These biological processes and activities play an essential role in regulating fat deposition, which may be one of the reasons why the IMF content of leg muscle is higher than that of breast muscle. Moreover, the subcellular localization of 190 differentially expressed proteins showed that they were mainly located in cytoplast (92 proteins, 48.42%), mitochondria (28 proteins, 14.74%), nucleus (24 proteins, 12.63%) and other subcells. Among them, mitochondria are the central place for conducting aerobic respiration in cells where 95% of the energy required for cell life activities comes from mitochondria, which contributes to the metabolism of substances and the deposition of fat. The analysis of 190 differential proteins by COG showed that only 100 of them could be identi ed. Among them, 43 were related to the synthesis and degradation of fat. They participated in energy production and conversion (15 proteins), carbohydrate transport and metabolism (15 proteins), lipid transport and metabolism (13 proteins), and other biological functions. Therefore, it should be noted that the difference in fat deposition between leg muscle and breast muscle is primarily due to the difference in the biological functions of essential proteins controlling the synthesis and degradation of fat.
Using KEGG to analyze the pathways of differentially expressed proteins, it was found that the proteins with signi cant differences were mainly enriched by pathways related to sugar metabolism and fat deposition such as ga00500 starch and sucrose metabolism, gga00071 fatty acid degradation, gga01212 fatty acid metabolism, and gga03320 PPAR signaling pathway. Hence, the differential protein may regulate the differential deposition of IMF in leg muscles and breast muscles through glucose metabolism, fat synthesis, and degradation. The results of the present study further interpreted the biological characteristics of IMF speci c deposition between Jingyuan chicken leg muscles and breast muscles.
PRM was used to quantify 190 proteins with differential expression, of which 23 of them were selected for PRM quanti cation. Due to the characteristics of certain proteins and the frequency of their expression, only 21 of them have been quanti ed. Further analysis found that among the 21 proteins quanti ed, ten proteins that regulate fat deposition have major variations in expression between leg muscles and breast muscles. For example, Acetyl CoA Acyltransferase 2 (AcetylCoAacyltransferase 2, ACAA2) is a key enzyme for fatty acid oxidation, widely distributed in mammalian and bird tissues and cytoplasm, and plays a vital role in the metabolism of fatty acids (Sodhi et al. 2014), and regulates intermuscular fat content (Miltiadou et al. 2017). Hence, this gene is considered a candidate for detecting intermuscular fat content (Miltiadou et al. 2017). In addition another study showed that the expression of the ACAA2 gene in chicken leg muscles was signi cantly higher than that of breast muscles (Liu Chen 2009).
Long-chain acyl-CoA dehydrogenase (Acyl-CoAdehydroge-nase, long-chain ACADL) is a coenzyme of acetylase in the body that plays an important role in the metabolism of carbohydrates, proteins, and lipids, particularly in long-chain β-oxidation fatty acids, and its regulation mechanism mainly includes regulations of expression and activity, has a very close relationship ( . Studies have shown that the expression of the ECI1 gene in Chinese pig back has the longest muscle is signi cantly higher than that of other foreign lean meat pigs, indicating that it is likely to be the primary gene-regulating pig fat metabolism (Lu Yunfeng et al. 2015).
Heart fatty acid-binding protein (FABP3 or H-FABP) is a member of the fatty acid-binding protein family, which expressed in the myocardium and skeletal muscle (Veerkamp & Maatman 1995). It is involved in maintaining the energy balance of the heart and other body tissues, regulating muscle fat content, and By analyzing ten differential proteins, we found that these differential proteins are signi cantly related to fat deposition, and demonstrate that regulating fat deposition is not a simple biological process that a single protein or gene can complete. So far, no reports are available regarding the mechanism of speci c deposition of IMF in different parts of Jingyuan chicken. We used a mathematical program (STRING and Cytoscape software) to perform the network interactions on ten screened proteins and mapped between them. We found that only eight proteins among ten have participated in network interaction, constituting a total of 19 relatively complex network pathways.

Conclusion
To conclude, we used label-free proteomics and PRM methods to carry out basic research on the leg and breast muscles of Jingyuan chicken. This study not only explains the molecular mechanism of IMF speci c deposition between the leg muscles and the breast muscles of Jingyuan chicken but also analyzes the network interaction of essential functional proteins regulating IMF deposition. In this study, we conclude that the key proteins regulate the synthesis and degradation of fat in different parts of the body through biological processes. The value and signi cance of these results are mainly re ected in that they not only provide molecular materials for the study of increasing IMF content of broilers but also establish an ideal animal model for the future research of meat quality. Hence, the present research has typical biological signi cance for the molecular breeding of high-quality local broilers.  Note: TJ stands for leg muscles and XJ stands for pectoral muscles. Location map of differentially expressed proteins of Jingyuan chicken leg muscle and pectoral muscle in subcellular structure. Note: TJ stands for leg muscles and XJ stands for pectoral muscles.

Abbreviations
Page 25/27  Enrichment and distribution map of differentially expressed proteins of Jingyuan chicken leg muscle and pectoral muscle in GO functional classi cation. Note: TJ stands for leg muscles and XJ stands for pectoral muscles.