In vitro protein expression profile of cultivated muscle cells from 
The growth of muscle in fish is continuous throughout their lives, unlike other vertebrates, thus, making fish a good model for studying cultivated meat production. Fish muscle also exhibits hypertrophy as well as hyperplasia , which provides further utility to understanding muscle cell growth. To conduct cell-based food research, the development of fish muscle cell lines is necessary to gain a deeper understanding of growth and maturation into viable muscle tissues. Molecular mechanisms of muscle growth and differentiation in animals can be studied in vitro using cell lines . As teleost do not have continuous muscle cell lines, such studies are still in their infancy, except for Scophthalmus maximus , Danio rerio , and Wallago attu . Most of these cell lines have not been used for cultivated meat research. Recently, a continuous fish muscle cell line, “mack cells”, was developed from the Atlantic mackerel ( Scomber scombrus) providing insight into cultivated seafood research . Fish muscle mass accounts for 50–70% of its total body weight and thus, muscle tissue production is crucial for cell-cultivated fish meat. It is important to understand the underlying mechanisms controlling fish muscle growth with the growth hormone/insulin-like growth factor, GH-IGF system, to improve the quality and quantity of myotomal muscle formation in vitro and in vivo. In vitro studies of fish cells and tissues have not been widely conducted for cell-based meat research . Beyond transcriptional regulatory factors, the analysis of proteome profiles is required to comprehend the molecular mechanisms behind phenotypic change during myogenic growth and differentiation. Both primary muscle cultures and established cell lines , have been studied using proteomic methods to gain insight into the protein groups expressed during the formation of myotubes. Knowledge of the proteome complements gene expression profiles during myogenesis, helping understand how different biochemical processes are coordinated at the cellular level . A key goal for the field of cultivated meat is to control the texture or quality of the tissue by adjusting various growth conditions in the cultured cells to regulate the myogenic differentiation through proteome approach. Depending on the type of muscle, stage of development, and physiological conditions, the differential expression in the proteome profile can be observed . The proteome of muscle cells is highly specialised due to the specific tasks of muscle cells. Proteomics has been used in cultivated meat research to improve cell culture strategies. For example, oxidative stress was identified as a contributing factor in the loss of stemness in porcine muscle cells; thus, L-ascorbic acid 2-phosphate (Asc-2P) was added to the cell culture medium . This improved cell proliferation and preserved muscle cell differentiation . The proteome expression of muscle cells in Labeo rohita has not been studied. It is crucial to generate and characterise fish cell lines based on muscle in order to promote meat-like tissues as well as for cell-based aquaculture research. Native muscle tissues from freshwater and marine species have been the subject of several experiments , . In view of the growing importance of cell-cultivated muscle tissues, the current study was aimed at a proteomics assessment from a muscle cell line from Labeo rohita . 

Cell culture The muscle explants of L. rohita adhered in flasks after media addition. The radiation of cells started after 120 h of explant preparation (Fig. a) and the first subculture was completed after 12 days. Subsequent passage was performed every 3 days with a split ratio of 1:2 (Fig. b and c). The LR muscle cells were maintained in an L-15 medium supplemented with 15% FBS for the first five subcultures and the serum concentration was reduced to 10% for subsequent passages. The LR muscle cells were successfully maintained up to 25 passages (Fig. d). Proteome characterization of LR muscle cells The total protein was extracted from the LR muscle cells at subcultures 1 (T1), 15 (T2), and 25 (T3). The concentration of the extracted protein was determined by Bradford assay and the protein quality was assessed using SDS-PAGE (Fig. ). A total of 300 proteins were obtained after MaxQuant analysis and further proteins were selected based on ≥ 2 unique peptides. A Venn diagram displaying the number of proteins identified between three passages and proteins that overlap between groups (Fig. ). The differentially expressed proteins across the different passages are represented in Tables S1, S2, and S3. A total of 11 unique proteins were observed in passage 1 (T1) and two unique proteins were observed in both passages 15 (T2) and 25 (T3) (Table S4). Proteins were filtered for those containing at least 2 unique peptides. Differential expression was performed in a pairwise manner for the 3 passages. In total, 120 unique proteins were differentially expressed in all the conditions. Hierarchical clustering of the top 25 proteins with significantly expressed of proteins (Student t-test, p < 0.05) was observed in triplicates (R1, R2, R3) across T1 and T2, T2 and T3, and T1 and T3, respectively. The differentially expressed proteins with adjusted p values less than 0.05 and fold-change greater than 1.5 are represented in the volcano plot and the top six expressed proteins (three upregulated and three downregulated) are represented in bar plots across the T1 and T2 (Fig. ), T2 and T3 (Fig. ) and T1 and T3 (Fig. ) groups, respectively. Protein–Protein interaction networks of the differentially expressed proteins with respective KEGG pathways are represented (STRING) for T1 and T2 (Fig. ) and T1 and T3 (Fig. ) groups, respectively. The functional annotation of protein-encoding genes was obtained from metascape and significantly enriched protein in terms of gene ontology for different passage group LR muscle cells were observed (Fig. ). In metascape, different pathways have been mapped for differentially expressed proteins for LR muscle cells at different passages (Table S5). 

The muscle explants of L. rohita adhered in flasks after media addition. The radiation of cells started after 120 h of explant preparation (Fig. a) and the first subculture was completed after 12 days. Subsequent passage was performed every 3 days with a split ratio of 1:2 (Fig. b and c). The LR muscle cells were maintained in an L-15 medium supplemented with 15% FBS for the first five subcultures and the serum concentration was reduced to 10% for subsequent passages. The LR muscle cells were successfully maintained up to 25 passages (Fig. d). 

The total protein was extracted from the LR muscle cells at subcultures 1 (T1), 15 (T2), and 25 (T3). The concentration of the extracted protein was determined by Bradford assay and the protein quality was assessed using SDS-PAGE (Fig. ). A total of 300 proteins were obtained after MaxQuant analysis and further proteins were selected based on ≥ 2 unique peptides. A Venn diagram displaying the number of proteins identified between three passages and proteins that overlap between groups (Fig. ). The differentially expressed proteins across the different passages are represented in Tables S1, S2, and S3. A total of 11 unique proteins were observed in passage 1 (T1) and two unique proteins were observed in both passages 15 (T2) and 25 (T3) (Table S4). Proteins were filtered for those containing at least 2 unique peptides. Differential expression was performed in a pairwise manner for the 3 passages. In total, 120 unique proteins were differentially expressed in all the conditions. Hierarchical clustering of the top 25 proteins with significantly expressed of proteins (Student t-test, p < 0.05) was observed in triplicates (R1, R2, R3) across T1 and T2, T2 and T3, and T1 and T3, respectively. The differentially expressed proteins with adjusted p values less than 0.05 and fold-change greater than 1.5 are represented in the volcano plot and the top six expressed proteins (three upregulated and three downregulated) are represented in bar plots across the T1 and T2 (Fig. ), T2 and T3 (Fig. ) and T1 and T3 (Fig. ) groups, respectively. Protein–Protein interaction networks of the differentially expressed proteins with respective KEGG pathways are represented (STRING) for T1 and T2 (Fig. ) and T1 and T3 (Fig. ) groups, respectively. The functional annotation of protein-encoding genes was obtained from metascape and significantly enriched protein in terms of gene ontology for different passage group LR muscle cells were observed (Fig. ). In metascape, different pathways have been mapped for differentially expressed proteins for LR muscle cells at different passages (Table S5). 

The current study was carried out to develop proteome characterization of a muscle cell line from L. rohita. Rohu is a commercially important and widely cultured freshwater species in south Asia. The process of cell line development started with primary culture through explantation. The explantation technique has several advantages over trypsinization in terms of maintenance, speed, ease, and avoids cell damage from enzymatic digestion . The cell line developed from Cirrhinus mrigala by the explant method showed better adhesion of tissue to the culture flask . The primary culture of muscle cells constituted a heterogenous group of cells with domination by fibroblast-like cells after the 5th passage. Similar findings were found with muscle cells of Scophthalmus maximus L. and Cromileptes altivelis . The culture medium for cell growth and proliferation plays a crucial role in cell line development and Leibovitz-15 is the most preferred cultured medium for fish cells – . Proteome characterization of LR muscle cells The proteomic profile of LR muscle cells at different passages was investigated to understand the molecular mechanisms underlying transcription factors during passaging. In previous 2DE-based proteomics, a total of 132 proteins were differentially expressed in gilthead sea bream primary cells with different passages . The differentially expressed proteins were analysed with adjusted p values less than 0.05 and fold-change of 1.5. The protein–protein interaction network of differentially expressed proteins with respective KEGG pathways were represented based on STRING. The functional annotation of protein-coding genes was obtained from metascape data. The whole proteome of rohu was built for aquaculture and protocols optimized for various tissues to obtain large-scale proteins for biological study – . Differentially expressed proteins across passage 1 (T1) and passage 15 (T2) Haemoglobin subunit alpha-like protein (hba1), striated muscle-like isoform X2, and ribosomal protein S8 (rps8a) are very abundant across T1 and T2. Alpha-globin is a part (subunit) of haemoglobin, a protein found in red blood cells that transports oxygen to cells and tissues. A relatively abundant haemoglobin subunit was identified in the earlier passages, which may engage in supplying cells with oxygen. A balanced metabolism, movement, and the provision of oxygen required for the entire body depend on striated muscles. Rps8 was one of 300 genes that were elevated in the whole trout between 4 and 13 weeks post-spawning that were enriched with genes that encode ribosomal subunits or regulate protein folding . In our study, rps8a was upregulated in the T1, involved in the growth stage and controlling protein production and maturation. Proteins such as ribosomal protein L30 (rpl30), apolipo A-I (apoa1), and carbonic anhydrase (cahz) were downregulated in T1 compared to T2 passages. For normal cellular homeostasis, carbonic anhydrase controls the acid–base balance in cells. High-density lipoprotein (HDL), aids in the transportation of phospholipids and cholesterol and is also known to carry the genes for intramuscular adipocytes. In earlier studies, L-15 supplemented with 1 g/L glucose stimulated adipogenesis in human normal (HNPA) and lipoma-derived (HLPA) pre-adipocytes . In the present study, L-15 media was employed to develop the muscle cell culture system of rohu by keeping neutral pH in medium supported proliferate and differentiation process. Myocytes and preadipocytes were isolated and cultured in mackerel, salmon, and trout using L-15 medium , , . The proteomics of breast muscle in regional breeds of chicken identified APOA1 as a potential candidate for intramuscular fat deposition . Rpl30 was found in trout muscle after 4 to 13 weeks after spawning , and rpl30 was downregulated in the current study between T1 and T2. Differentially expressed proteins across passage 15 (T2) and passage 25 (T3) In comparing T2 and T3, there were elevated levels of lactate dehydrogenase (ldha), LIM domain-binding 3-like protein (ldb1a), and Rho guanine nucleotide exchange factor 9 (arhgef9a) in T2. Ldha activity increased linearly as myoblasts proliferated. However, Barjot et al. (1998) observed no significant difference in the expression of ldha during early stages of rabbit embryonic muscle cell cultures . Muscle LIM protein (MLP), a LIM-only factor unique to muscles, has two distinct subcellular localizations and was found in both the nucleus and the cytoplasm of cells. In C2C12 myoblasts, MLP overexpression promotes skeletal myogenesis, whereas MLP inhibition prevents terminal differentiation . By activating the Rho GTPases, Rho family guanine nucleotide exchange factors (GEFs) control a variety of cellular activities, such as cytoskeletal reorganization, cell adhesion, and differentiation. The development of numerous cell types, such as neurons, T lymphocytes, myocytes, adipocytes, and keratinocytes, was expressed by Rho family proteins . Significantly downregulated proteins in our study were Dihydrolipoyl dehydrogenase (dld), Tropomyosin Alpha-3 Chain Isoform X6 (tpm3), and Rho GDP-dissociation Inhibitor 1 (arhgdia). These key regulatory enzymes in pyruvate dihydrolipoamide dehydrogenase (DLD) were lower in T2 than T3 passage and are crucial for cellular energy production. In vitro, myogenesis has been associated with increased synthesis and accumulation of tropomyosin . The tropomyosin genes are constitutively expressed during muscle development . In bovine satellite cells (bovine semitendinosus muscle), Rho GDP-dissociation inhibitor 1 is implicated in myogenesis and is elevated during myogenesis . The myogeneis has previously been shown to be influenced by a number of up-regulated proteins, including annexin 2, Rho GDP-dissociation inhibitor 1 (Rho GDI), and LIM domain-containing protein in bovine satellite cells . Differentially expressed proteins across passage 1 (T1) and passage 25 (T3) Myosin-binding H-like protein, vesicle-fusing ATPase (bnsfb), and aconitate hydratase, mitochondrial (aco2) were upregulated in T1 compared to T3. MyBP-H is part of the thick filaments in skeletal muscle and has a great affinity for myosin and serves as a biomarker for the dysregulation of actin-myosin interaction in skeletal muscle . Based on binding of myosin and how the motor protein is organized inside the thick filament, MyBP-H plays a crucial part in myofibrillogenesis and may serve as an early indicator of muscle regeneration . Early passages were composed of more myofibrillar proteins than the T3 passages, suggesting a decrease in myogenic stemness over time. To fuse myoblasts and repair damaged membranes, vesicle transport is necessary, and rearranging the actin cytoskeleton is necessary for the movement of vesicles – . Additionally, it was demonstrated that ATP and PIP2 were essential for effective myoblast fusion at the membrane . In the present study, the early stages of muscle cells showed the myoblast fusion in the T1 group. The proteins of the T1 group were substantially more active and involved in metabolism at a higher rate than the proteins of the T2 groups. Reactive oxygen species (ROS) deactivate the mitochondrial enzyme aconitase, which is involved in the tricarboxylic acid cycle and transforms citrate to isocitrate. At least two processes, phosphorylation/dephosphorylation and oxidation/reduction, can control aconitase activity . Thioredoxin-dependent peroxide mitochondrial, Annexin (anxa13l), and ADP-ribosylation factor (arf1) proteins were significantly downregulated across the T1 and T3 groups. One of the main drivers of adaptive alterations in contracting skeletal muscle has been identified as reactive oxygen species (ROS), especially hydrogen peroxide (H 2 O 2 ). In rat studies, boosting phosphatidylinositol 3-kinase/protein kinase B/forkhead Box O1 signaling, by treatment with recombinant human Trx, reduced H 2 O 2 -induced oxidative stress and increased myogenic differentiation of rat BMSCs . Although the cells in our case were healthier and there was no strong expression in the first passage, this protein is essential for cells in myotube or myoblast stages and was expressed at passage T3. Molecular and imaging methods in mouse myoblasts demonstrated that ANXA1 contributes to C2C12 cell differentiation . The ANXA1 expression rises during differentiation and its downregulation sharply reduces differentiation . Although it has not yet been investigated, it has been suggested that ADP-ribosylation controls differentiation and dedifferentiation. Protein–protein interaction network across three passages of LR muscle cells Protein–Protein interactions of significantly expressed proteins between T1 and T2 groups were Histone-lysine N-methyltransferase SMYD1-like protein (smyd1), Phosphoglycerate mutase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and enolase. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and annexin (ANXA1) are proteins identified as overexpressed in microtubule formation in an earlier investigation using 2DE proteomics . As a result, the development of the microtrabecular lattices was dependent on GAPDH interactions with microtubules , . Enolase and glycogen phosphorylase are two examples of glycolysis-related proteins. These proteins confirm the transition from proliferation to differentiation, which is marked by an increase in the glycolytic machinery, and was previously observed during early myogenesis in cows and the proteome of gilthead sea bream comparing 4 and 8 days of myogenesis. The eukaryotic translation initiation factor 5A (eIF5A) was significantly lower in the T1 groups and is an evolutionarily conserved protein that binds ribosomes to facilitate the translation of peptide motifs with consecutive prolines. Ribosomal protein L27 (rpl27), Adenylate kinase isoenzyme 1 (ak1), and Pyruvate dehydrogenase E1 component subunit beta (pdhb) proteins were upregulated across T1 and T3. In the early phase of LR muscle cells, ribosomal activity in T1 contributed to higher rates of protein synthesis. In an earlier study, controlling protein synthesis and maturation for cell growth, cluster III genes in trout became dominant in the period between 4 and 13 weeks following spawning . PDHB increased during mouse and primary human muscle cell differentiation and encouraged myogenic differentiation . The regulation of Rpl27 and pdhb were observed in the present study and reduced from T1 to T3. In comparison to the early stage, PDHB expression was reduced in older mouse muscle (− 50% of mRNA level, P 0.01) . With the high levels of adenylate kinase 1 (AK1) and ATP synthase during C2C12 myogenesis, extracellular ATP production was significantly boosted. The protein ak1 increased and is involved in the process of myogenesis . Since myotubes exhibit a significant increase in AK1-dependent exATP synthesis, it is tempting to assume that exATP is necessary for myogenesis, which has been shown to be robustly stimulated by exATP exposure . Malate dehydrogenase (mdh2), ribosomal protein L3 (rpl3), and fructose-bisphosphate aldolase (aldob) were significantly lower between T1 and T3. Two important glyceroneogenic enzymes, cytoplasmic malate dehydrogenase (MDHC) and cytosolic phosphoenolpyruvate carboxykinase (PEPCKC), have been shown to be upregulated during terminal differentiation of adipogenesis . The ability of ribosome specialization in skeletal muscle and its function in controlling skeletal muscle growth is supported by RPL3L expression. In response to a hypertrophic stimulus, Rpl3 expression was concurrently up-regulated while Rpl3 expression was significantly down-regulated, indicating that Rpl3l may play a role in controlling skeletal muscle mass . Fructose-l,6-bisphosphatase (FBPase) was present in both the cytoplasm and the nuclei of the cells during the proliferative phase that was characterized by the expression of MyoD. Aldob levels were lower in T1 than in T2 or T3 in the present study, and this finding was supported by the reduction of expression of MyoD in the T1 group. Pathways enriched during different passages of LR muscle cells Glycolysis, metabolic, and carbon metabolism in the muscle proliferation process In the present study, glycolytic proteins were upregulated in the T1 group compared to T2 and T3. Proteins involved in actin cytoskeleton remodelling and cellular metabolic functions (such as transcription, ubiquitination, stress response, and glucose metabolism) were enriched during the proliferation process. In the seabream primary culture with proteome profiling performed at passages 3, 8, and 12, the 40 differentially expressed proteins were connected to a variety of functional categories related to cell structure or cell metabolism . During embryonic development in zebrafish, glycolysis was an important pathway in myoblast fusion . Pgam2 was upregulated in the early stage of the passage indicating the presence of fast-growing phenotypes in LR muscle cells early passages. Similarly, pgam2 in zebrafish morphants was observed and suggested a strongly conserved role in myoblast fusion-based muscle growth in glycolysis . Cytoskeleton, ribosomal and carbon metabolism involved in muscle differentiation The commencement of differentiation was characterized by a gradual rise in the number of proteins involved in cytoskeleton reorganization and adhesion processes. The actin cytoskeleton, which is essential for cell migration, includes cytoskeletal proteins regulated by ANXA1. Annexin expression was observed at all passages, while reduced in the initial passage in comparison to passage 25, indicating more cell differentiation higher in later passages. In a previous study, annexin expression varied during cell proliferation, differentiation and developmental stage . The present results suggest that the reduction of annexin in early passages may play a role in proliferation and in later passages involved in myotube formation as part of the differentiation process. The commencement of differentiation was characterized by a gradual rise in the number of proteins involved in cytoskeleton reorganization and adhesion processes. The Smyd family is essential for the formation of sarcomeres during myofibrillogenesis . Smyd1 was initially localized in the nucleus of C2C12 myoblasts and then moves from the nucleus to the cytoplasm as myoblasts differentiate into myotubes . Compared to T2, the initial T1 passage contained upregulated cytoskeleton proteins involved in the myoblast or differentiation process. Ribosome biogenesis has emerged as an important regulator of skeletal muscle growth and maintenance by altering the translational capacity of the cell. During the translation process in the gilthead sea bream, rps12 was responsible in the selection of tRNA in an efficient manner and similar to rps12 found , . The evolutionarily conserved protein known as the eukaryotic translation initiation factor 5A (eIF5A) interacts with ribosomes to aid in the translation of peptide motifs containing successive prolines or prolines joined to glycine and charged amino acids. In carbon metabolism, the got1 gene was closely linked to cell proliferation and glutamate synthesis . 

The proteomic profile of LR muscle cells at different passages was investigated to understand the molecular mechanisms underlying transcription factors during passaging. In previous 2DE-based proteomics, a total of 132 proteins were differentially expressed in gilthead sea bream primary cells with different passages . The differentially expressed proteins were analysed with adjusted p values less than 0.05 and fold-change of 1.5. The protein–protein interaction network of differentially expressed proteins with respective KEGG pathways were represented based on STRING. The functional annotation of protein-coding genes was obtained from metascape data. The whole proteome of rohu was built for aquaculture and protocols optimized for various tissues to obtain large-scale proteins for biological study – . 

Haemoglobin subunit alpha-like protein (hba1), striated muscle-like isoform X2, and ribosomal protein S8 (rps8a) are very abundant across T1 and T2. Alpha-globin is a part (subunit) of haemoglobin, a protein found in red blood cells that transports oxygen to cells and tissues. A relatively abundant haemoglobin subunit was identified in the earlier passages, which may engage in supplying cells with oxygen. A balanced metabolism, movement, and the provision of oxygen required for the entire body depend on striated muscles. Rps8 was one of 300 genes that were elevated in the whole trout between 4 and 13 weeks post-spawning that were enriched with genes that encode ribosomal subunits or regulate protein folding . In our study, rps8a was upregulated in the T1, involved in the growth stage and controlling protein production and maturation. Proteins such as ribosomal protein L30 (rpl30), apolipo A-I (apoa1), and carbonic anhydrase (cahz) were downregulated in T1 compared to T2 passages. For normal cellular homeostasis, carbonic anhydrase controls the acid–base balance in cells. High-density lipoprotein (HDL), aids in the transportation of phospholipids and cholesterol and is also known to carry the genes for intramuscular adipocytes. In earlier studies, L-15 supplemented with 1 g/L glucose stimulated adipogenesis in human normal (HNPA) and lipoma-derived (HLPA) pre-adipocytes . In the present study, L-15 media was employed to develop the muscle cell culture system of rohu by keeping neutral pH in medium supported proliferate and differentiation process. Myocytes and preadipocytes were isolated and cultured in mackerel, salmon, and trout using L-15 medium , , . The proteomics of breast muscle in regional breeds of chicken identified APOA1 as a potential candidate for intramuscular fat deposition . Rpl30 was found in trout muscle after 4 to 13 weeks after spawning , and rpl30 was downregulated in the current study between T1 and T2. 

In comparing T2 and T3, there were elevated levels of lactate dehydrogenase (ldha), LIM domain-binding 3-like protein (ldb1a), and Rho guanine nucleotide exchange factor 9 (arhgef9a) in T2. Ldha activity increased linearly as myoblasts proliferated. However, Barjot et al. (1998) observed no significant difference in the expression of ldha during early stages of rabbit embryonic muscle cell cultures . Muscle LIM protein (MLP), a LIM-only factor unique to muscles, has two distinct subcellular localizations and was found in both the nucleus and the cytoplasm of cells. In C2C12 myoblasts, MLP overexpression promotes skeletal myogenesis, whereas MLP inhibition prevents terminal differentiation . By activating the Rho GTPases, Rho family guanine nucleotide exchange factors (GEFs) control a variety of cellular activities, such as cytoskeletal reorganization, cell adhesion, and differentiation. The development of numerous cell types, such as neurons, T lymphocytes, myocytes, adipocytes, and keratinocytes, was expressed by Rho family proteins . Significantly downregulated proteins in our study were Dihydrolipoyl dehydrogenase (dld), Tropomyosin Alpha-3 Chain Isoform X6 (tpm3), and Rho GDP-dissociation Inhibitor 1 (arhgdia). These key regulatory enzymes in pyruvate dihydrolipoamide dehydrogenase (DLD) were lower in T2 than T3 passage and are crucial for cellular energy production. In vitro, myogenesis has been associated with increased synthesis and accumulation of tropomyosin . The tropomyosin genes are constitutively expressed during muscle development . In bovine satellite cells (bovine semitendinosus muscle), Rho GDP-dissociation inhibitor 1 is implicated in myogenesis and is elevated during myogenesis . The myogeneis has previously been shown to be influenced by a number of up-regulated proteins, including annexin 2, Rho GDP-dissociation inhibitor 1 (Rho GDI), and LIM domain-containing protein in bovine satellite cells . 

Myosin-binding H-like protein, vesicle-fusing ATPase (bnsfb), and aconitate hydratase, mitochondrial (aco2) were upregulated in T1 compared to T3. MyBP-H is part of the thick filaments in skeletal muscle and has a great affinity for myosin and serves as a biomarker for the dysregulation of actin-myosin interaction in skeletal muscle . Based on binding of myosin and how the motor protein is organized inside the thick filament, MyBP-H plays a crucial part in myofibrillogenesis and may serve as an early indicator of muscle regeneration . Early passages were composed of more myofibrillar proteins than the T3 passages, suggesting a decrease in myogenic stemness over time. To fuse myoblasts and repair damaged membranes, vesicle transport is necessary, and rearranging the actin cytoskeleton is necessary for the movement of vesicles – . Additionally, it was demonstrated that ATP and PIP2 were essential for effective myoblast fusion at the membrane . In the present study, the early stages of muscle cells showed the myoblast fusion in the T1 group. The proteins of the T1 group were substantially more active and involved in metabolism at a higher rate than the proteins of the T2 groups. Reactive oxygen species (ROS) deactivate the mitochondrial enzyme aconitase, which is involved in the tricarboxylic acid cycle and transforms citrate to isocitrate. At least two processes, phosphorylation/dephosphorylation and oxidation/reduction, can control aconitase activity . Thioredoxin-dependent peroxide mitochondrial, Annexin (anxa13l), and ADP-ribosylation factor (arf1) proteins were significantly downregulated across the T1 and T3 groups. One of the main drivers of adaptive alterations in contracting skeletal muscle has been identified as reactive oxygen species (ROS), especially hydrogen peroxide (H 2 O 2 ). In rat studies, boosting phosphatidylinositol 3-kinase/protein kinase B/forkhead Box O1 signaling, by treatment with recombinant human Trx, reduced H 2 O 2 -induced oxidative stress and increased myogenic differentiation of rat BMSCs . Although the cells in our case were healthier and there was no strong expression in the first passage, this protein is essential for cells in myotube or myoblast stages and was expressed at passage T3. Molecular and imaging methods in mouse myoblasts demonstrated that ANXA1 contributes to C2C12 cell differentiation . The ANXA1 expression rises during differentiation and its downregulation sharply reduces differentiation . Although it has not yet been investigated, it has been suggested that ADP-ribosylation controls differentiation and dedifferentiation. 

Protein–Protein interactions of significantly expressed proteins between T1 and T2 groups were Histone-lysine N-methyltransferase SMYD1-like protein (smyd1), Phosphoglycerate mutase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and enolase. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and annexin (ANXA1) are proteins identified as overexpressed in microtubule formation in an earlier investigation using 2DE proteomics . As a result, the development of the microtrabecular lattices was dependent on GAPDH interactions with microtubules , . Enolase and glycogen phosphorylase are two examples of glycolysis-related proteins. These proteins confirm the transition from proliferation to differentiation, which is marked by an increase in the glycolytic machinery, and was previously observed during early myogenesis in cows and the proteome of gilthead sea bream comparing 4 and 8 days of myogenesis. The eukaryotic translation initiation factor 5A (eIF5A) was significantly lower in the T1 groups and is an evolutionarily conserved protein that binds ribosomes to facilitate the translation of peptide motifs with consecutive prolines. Ribosomal protein L27 (rpl27), Adenylate kinase isoenzyme 1 (ak1), and Pyruvate dehydrogenase E1 component subunit beta (pdhb) proteins were upregulated across T1 and T3. In the early phase of LR muscle cells, ribosomal activity in T1 contributed to higher rates of protein synthesis. In an earlier study, controlling protein synthesis and maturation for cell growth, cluster III genes in trout became dominant in the period between 4 and 13 weeks following spawning . PDHB increased during mouse and primary human muscle cell differentiation and encouraged myogenic differentiation . The regulation of Rpl27 and pdhb were observed in the present study and reduced from T1 to T3. In comparison to the early stage, PDHB expression was reduced in older mouse muscle (− 50% of mRNA level, P 0.01) . With the high levels of adenylate kinase 1 (AK1) and ATP synthase during C2C12 myogenesis, extracellular ATP production was significantly boosted. The protein ak1 increased and is involved in the process of myogenesis . Since myotubes exhibit a significant increase in AK1-dependent exATP synthesis, it is tempting to assume that exATP is necessary for myogenesis, which has been shown to be robustly stimulated by exATP exposure . Malate dehydrogenase (mdh2), ribosomal protein L3 (rpl3), and fructose-bisphosphate aldolase (aldob) were significantly lower between T1 and T3. Two important glyceroneogenic enzymes, cytoplasmic malate dehydrogenase (MDHC) and cytosolic phosphoenolpyruvate carboxykinase (PEPCKC), have been shown to be upregulated during terminal differentiation of adipogenesis . The ability of ribosome specialization in skeletal muscle and its function in controlling skeletal muscle growth is supported by RPL3L expression. In response to a hypertrophic stimulus, Rpl3 expression was concurrently up-regulated while Rpl3 expression was significantly down-regulated, indicating that Rpl3l may play a role in controlling skeletal muscle mass . Fructose-l,6-bisphosphatase (FBPase) was present in both the cytoplasm and the nuclei of the cells during the proliferative phase that was characterized by the expression of MyoD. Aldob levels were lower in T1 than in T2 or T3 in the present study, and this finding was supported by the reduction of expression of MyoD in the T1 group. 

Glycolysis, metabolic, and carbon metabolism in the muscle proliferation process In the present study, glycolytic proteins were upregulated in the T1 group compared to T2 and T3. Proteins involved in actin cytoskeleton remodelling and cellular metabolic functions (such as transcription, ubiquitination, stress response, and glucose metabolism) were enriched during the proliferation process. In the seabream primary culture with proteome profiling performed at passages 3, 8, and 12, the 40 differentially expressed proteins were connected to a variety of functional categories related to cell structure or cell metabolism . During embryonic development in zebrafish, glycolysis was an important pathway in myoblast fusion . Pgam2 was upregulated in the early stage of the passage indicating the presence of fast-growing phenotypes in LR muscle cells early passages. Similarly, pgam2 in zebrafish morphants was observed and suggested a strongly conserved role in myoblast fusion-based muscle growth in glycolysis . Cytoskeleton, ribosomal and carbon metabolism involved in muscle differentiation The commencement of differentiation was characterized by a gradual rise in the number of proteins involved in cytoskeleton reorganization and adhesion processes. The actin cytoskeleton, which is essential for cell migration, includes cytoskeletal proteins regulated by ANXA1. Annexin expression was observed at all passages, while reduced in the initial passage in comparison to passage 25, indicating more cell differentiation higher in later passages. In a previous study, annexin expression varied during cell proliferation, differentiation and developmental stage . The present results suggest that the reduction of annexin in early passages may play a role in proliferation and in later passages involved in myotube formation as part of the differentiation process. The commencement of differentiation was characterized by a gradual rise in the number of proteins involved in cytoskeleton reorganization and adhesion processes. The Smyd family is essential for the formation of sarcomeres during myofibrillogenesis . Smyd1 was initially localized in the nucleus of C2C12 myoblasts and then moves from the nucleus to the cytoplasm as myoblasts differentiate into myotubes . Compared to T2, the initial T1 passage contained upregulated cytoskeleton proteins involved in the myoblast or differentiation process. Ribosome biogenesis has emerged as an important regulator of skeletal muscle growth and maintenance by altering the translational capacity of the cell. During the translation process in the gilthead sea bream, rps12 was responsible in the selection of tRNA in an efficient manner and similar to rps12 found , . The evolutionarily conserved protein known as the eukaryotic translation initiation factor 5A (eIF5A) interacts with ribosomes to aid in the translation of peptide motifs containing successive prolines or prolines joined to glycine and charged amino acids. In carbon metabolism, the got1 gene was closely linked to cell proliferation and glutamate synthesis . 

In the present study, glycolytic proteins were upregulated in the T1 group compared to T2 and T3. Proteins involved in actin cytoskeleton remodelling and cellular metabolic functions (such as transcription, ubiquitination, stress response, and glucose metabolism) were enriched during the proliferation process. In the seabream primary culture with proteome profiling performed at passages 3, 8, and 12, the 40 differentially expressed proteins were connected to a variety of functional categories related to cell structure or cell metabolism . During embryonic development in zebrafish, glycolysis was an important pathway in myoblast fusion . Pgam2 was upregulated in the early stage of the passage indicating the presence of fast-growing phenotypes in LR muscle cells early passages. Similarly, pgam2 in zebrafish morphants was observed and suggested a strongly conserved role in myoblast fusion-based muscle growth in glycolysis . 

The commencement of differentiation was characterized by a gradual rise in the number of proteins involved in cytoskeleton reorganization and adhesion processes. The actin cytoskeleton, which is essential for cell migration, includes cytoskeletal proteins regulated by ANXA1. Annexin expression was observed at all passages, while reduced in the initial passage in comparison to passage 25, indicating more cell differentiation higher in later passages. In a previous study, annexin expression varied during cell proliferation, differentiation and developmental stage . The present results suggest that the reduction of annexin in early passages may play a role in proliferation and in later passages involved in myotube formation as part of the differentiation process. The commencement of differentiation was characterized by a gradual rise in the number of proteins involved in cytoskeleton reorganization and adhesion processes. The Smyd family is essential for the formation of sarcomeres during myofibrillogenesis . Smyd1 was initially localized in the nucleus of C2C12 myoblasts and then moves from the nucleus to the cytoplasm as myoblasts differentiate into myotubes . Compared to T2, the initial T1 passage contained upregulated cytoskeleton proteins involved in the myoblast or differentiation process. Ribosome biogenesis has emerged as an important regulator of skeletal muscle growth and maintenance by altering the translational capacity of the cell. During the translation process in the gilthead sea bream, rps12 was responsible in the selection of tRNA in an efficient manner and similar to rps12 found , . The evolutionarily conserved protein known as the eukaryotic translation initiation factor 5A (eIF5A) interacts with ribosomes to aid in the translation of peptide motifs containing successive prolines or prolines joined to glycine and charged amino acids. In carbon metabolism, the got1 gene was closely linked to cell proliferation and glutamate synthesis . 

Experimental animal Live and healthy fingerlings of L. rohita (body weight: 15 to 20 g) were procured from Crescent Fisheries, Gujrat, and were transported to the Wet Laboratory, ICAR-Central Institute of Fisheries Education, Mumbai, India and maintained in deionized freshwater with suitable aeration. The donor fish were transported to the Fish Genetics and Biotechnology laboratory and kept for starvation in well-aerated sterile freshwater for 24 h before explant preparation to avoid the contamination from gut. Cell culture The starved fish were transported to the central cell culture facility and allowed to swim freely with well-aerated water containing surface disinfectant for 30 min to remove skin microflora. The disinfected fish were euthanized by exposure to rapid hypothermic shock in an ice-chilled bath for 1 min and then wiped with 70% ethanol. The muscle tissue was aseptically dissected using sterile scissors and placed in phosphate buffer saline (PBS) (Thermo Fisher Scientific). The washing steps were performed with PBS containing 500 IU/mL penicillin, 500 µg/mL streptomycin, and 2.5 μg/mL fungizone. The muscle explants were prepared by mincing a tissue to 1 mm 3 size in PBS and seeded in a cell culture flask (T-25). The excess PBS was removed and the adherence of explants was accomplished through the addition of 0.2 ml fetal bovine serum (FBS) (Gibco); the flasks were allowed for incubation at 28 °C overnight. The growth medium, Leibovitz-15 (L-15) (HiMedia) supplemented with 15% FBS was added to the flasks. The cells were subcultured once reached to 70–90% confluency. The medium was removed and the cells were harvested through trypsinization using 1 ml TPVG (0.25% trypsin) (HiMedia) solution. The harvested cells were immediately resuspended in a growth medium containing 15% serum and seeded into a 25 cm 2 (T-25) flask with a split ratio of 1:2. The flasks were incubated at 28 °C with optimum pH 7.4 and the subsequent subculture was performed once the cell confluency reached 90%. The serum concentration was reduced to 10% for subsequent passages. Proteome characterization of LR muscle cells Protein extraction from the LR muscle cells The confluent LR muscle cells at subcultures 1 (T1(R1, R2, R3)), 15 (T2 (R1, R2, R3)), and 25 (T3 (R1, R2, R3)) containing triplicate flasks for each replicates were harvested (2 × 10 6 ) using trypsin versene (Himedia, Mumbai) and the cells dislodged for 1 min. Harvested cells were homogenized in 500 µl of lysis buffer (7 M Urea, 2 M Thiourea, 4% CHAPS, 10 mM DTT, 5 µl protease inhibitor). Sonication was performed for 2 min with an amplitude of 40% and a 5-s pulse on and off. The tubes were centrifuged at 12,000 g for 10 min at 4 °C, and the supernatant was collected and incubated with 500 µL of 100% ice-chilled acetone (− 20 °C) overnight. Pellet formation was by centrifugation at 12,000 g for 10 min at 4 °C and the pellet was washed thrice with 80% chilled acetone. The resulting pellet was solubilized in the lysis buffer for 1–2 h in a vortex at room temperature. Centrifugation was performed for final protein extraction at 12,000 g for 10 min at 4 °C, and the supernatant was collected and stored at − 80 °C until further use. Protein quantification and digestion Cell lysates were processed for quantification of proteins using the Bradford method using bovine serum albumin as the standard. Briefly, five BSA standards were prepared at 10, 20, 30, 40, and 50 µg/ml and extracted samples were analyzed in a NanoDrop™ One/OneC Microvolume UV–Vis Spectrophotometer (Thermo Scientific). The in-solution-based digestion method was used to process 30 µg of protein. Prior to LC–MS/MS analysis, samples were desalted using an Empore C18 extraction column as per our earlier studies . Briefly, 30 µg of protein sample was employed in the C18 for each stage (200 μl pipette tips). A 40 μl of a 50% acetonitrile (ACN) solution was used to activate the desalting column. The liquid from the collection vial was centrifuged at 1000 g for 2 min at room temperature and discarded. The desalting column was cleaned with 40 μl of 99.9% ACN after the aforementioned step was repeated twice. Repeated centrifugation at 1000 g for 2 min at room temperature was used to remove the liquid from the collection vial. Formic acid (FA), solvent A, was used to equilibrate the desalting column twice using 40 μl of 0.1% (v/v) formic acid. Afterward, the centrifugation process was repeated and the liquid discarded. The samples were reconstituted in 40 μl of 0.1% (v/v) FA and vortexed slowly for 10 min. Reconstituted samples were spun at 1000 g for two minutes using a syringe or centrifuge to pass through the desalting column. This process was repeaed five times. A 50 μl sample of 40% (v/v) acetonitrile in 0.1% (v/v) FA was passed through the column and the elute was collected in a fresh 1.5 ml Eppendorf tube. The same vial was used to collect the elute after repeating the step with 50% and 60% (v/v) acetonitrile in 0.1% (v/v) FA. A 150 μl of the desalted sample was thereafter dried in a speed vacuum centrifuge. The desalted peptides were stored at -20 °C. Liquid chromatography-tandem mass spectrometry in data-dependent acquisition mode Peptides were measured for each sample (T1R1, T1R2, T1R3, T2R1, T2R2, T2R3, and T3R1, T3R2, T3R3) using the Scopes method for liquid chromatography-tandem mass spectrometric analysis (LC–MS/MS). On an Easy-nLC 1200 system, a 120-min gradient was applied to all samples. The peptide sample was placed into the trap column (100 mm × 2 cm, nanoViper C18, 5 mm, 100A; Thermo Fisher Scientific) at a flow rate of 5 μl/min. Peptides were separated on an analytical column (3 μm particle, and 100 Å pore size, 75 μm × 50 cm; Thermo Fisher Scientific) at a flow rate of 300 nl/min in solvent A (0.1% formic acid (FA) in water) and solvent B (80% acetonitrile with 0.1% FA) over a 120 min gradient). The mass spectrometry data were collected using an Orbitrap spectrometer connected to the aforementioned LC. The Orbitrap mass analyzer was used to perform mass spectrometric acquisition in data-dependent acquisition (DDA) mode over the entire scan range of 375–1700 m/z and with a mass resolution of 60,000. The mass window was set at 10 ppm with a dynamic exclusion time of 40 s. For fragmentation, all MS/MS spectra were obtained using the HCD method (High Energy Collision Dissociation). The AGC target for MS1 and MS2 was set at 400,000 and 10,000 respectively. A lock mass of 445.12003 m/z was used for positive internal calibration. MaxQuant analysis for proteome The raw mass spectrometry was analysed in MaxQuant (v1.6.6.0) software against the UniProt protein database for Labeo rohita using the in-built search engine, Andromeda. All raw files were analyzed together using Label-Free-Quantification (LFQ) parameters. Parameters include label type as standard with a multiplicity of 1 and the match between runs was checked. Orbitrap fusion mode was selected and trypsin as protease was used. A maximum of 2 missed cleavages were allowed. Under the fixed modification, carbamidomethylation at Cysteine (+ 57.021464 Da) and for variable modification, oxidation at Methionine (+ 15.994915 Da) was selected. Both at the protein and peptide levels, a false discovery level was kept at 1%. For the decoy mode option, the reverse was selected and proteins were identified with unique peptides only. For quantitative analysis, LFQ intensities obtained for each sample were selected. Statistical analysis using metaboanalyst Statistical analysis was performed in metaboanalyst across different passages of LR muscle cells T1 vs T2, T2 vs T3, and T1 vs T3 by taking MaxQuant analyzed files . The proteomic data of the nine samples were considered for further analysis. Missing values of the features having abundance values of more than 75% of each group were imputed by the KNN algorithm and considered for differential protein expression analysis. Log-transformed data along with normalization was taken for statistical analysis (i.e., t-test and fold change). Two sample t-test (Welch t-test) was performed to identify the significant differentially expressed proteins where a p value of 0.05 was set as the maximum threshold value. Among all t-test passed proteins, proteins with a fold change value of 1.5 were considered significantly differentially expressed proteins (DEPs). Pathway and protein–protein interaction enrichment analysis LFQ data of different passages were taken forward for functional annotation and biological pathway analysis. The gene ontology classification of dysregulated proteins was performed in the eggNOG (Version 6.0) . Later, the entire identified abundant proteome gene list was analyzed using Metascape between the passages . In addition, the network visualization and the protein–protein interaction analysis for selected pathways were done in STRING between the passages (version 11.0) . 

Live and healthy fingerlings of L. rohita (body weight: 15 to 20 g) were procured from Crescent Fisheries, Gujrat, and were transported to the Wet Laboratory, ICAR-Central Institute of Fisheries Education, Mumbai, India and maintained in deionized freshwater with suitable aeration. The donor fish were transported to the Fish Genetics and Biotechnology laboratory and kept for starvation in well-aerated sterile freshwater for 24 h before explant preparation to avoid the contamination from gut. 

The starved fish were transported to the central cell culture facility and allowed to swim freely with well-aerated water containing surface disinfectant for 30 min to remove skin microflora. The disinfected fish were euthanized by exposure to rapid hypothermic shock in an ice-chilled bath for 1 min and then wiped with 70% ethanol. The muscle tissue was aseptically dissected using sterile scissors and placed in phosphate buffer saline (PBS) (Thermo Fisher Scientific). The washing steps were performed with PBS containing 500 IU/mL penicillin, 500 µg/mL streptomycin, and 2.5 μg/mL fungizone. The muscle explants were prepared by mincing a tissue to 1 mm 3 size in PBS and seeded in a cell culture flask (T-25). The excess PBS was removed and the adherence of explants was accomplished through the addition of 0.2 ml fetal bovine serum (FBS) (Gibco); the flasks were allowed for incubation at 28 °C overnight. The growth medium, Leibovitz-15 (L-15) (HiMedia) supplemented with 15% FBS was added to the flasks. The cells were subcultured once reached to 70–90% confluency. The medium was removed and the cells were harvested through trypsinization using 1 ml TPVG (0.25% trypsin) (HiMedia) solution. The harvested cells were immediately resuspended in a growth medium containing 15% serum and seeded into a 25 cm 2 (T-25) flask with a split ratio of 1:2. The flasks were incubated at 28 °C with optimum pH 7.4 and the subsequent subculture was performed once the cell confluency reached 90%. The serum concentration was reduced to 10% for subsequent passages. 

Protein extraction from the LR muscle cells The confluent LR muscle cells at subcultures 1 (T1(R1, R2, R3)), 15 (T2 (R1, R2, R3)), and 25 (T3 (R1, R2, R3)) containing triplicate flasks for each replicates were harvested (2 × 10 6 ) using trypsin versene (Himedia, Mumbai) and the cells dislodged for 1 min. Harvested cells were homogenized in 500 µl of lysis buffer (7 M Urea, 2 M Thiourea, 4% CHAPS, 10 mM DTT, 5 µl protease inhibitor). Sonication was performed for 2 min with an amplitude of 40% and a 5-s pulse on and off. The tubes were centrifuged at 12,000 g for 10 min at 4 °C, and the supernatant was collected and incubated with 500 µL of 100% ice-chilled acetone (− 20 °C) overnight. Pellet formation was by centrifugation at 12,000 g for 10 min at 4 °C and the pellet was washed thrice with 80% chilled acetone. The resulting pellet was solubilized in the lysis buffer for 1–2 h in a vortex at room temperature. Centrifugation was performed for final protein extraction at 12,000 g for 10 min at 4 °C, and the supernatant was collected and stored at − 80 °C until further use. Protein quantification and digestion Cell lysates were processed for quantification of proteins using the Bradford method using bovine serum albumin as the standard. Briefly, five BSA standards were prepared at 10, 20, 30, 40, and 50 µg/ml and extracted samples were analyzed in a NanoDrop™ One/OneC Microvolume UV–Vis Spectrophotometer (Thermo Scientific). The in-solution-based digestion method was used to process 30 µg of protein. Prior to LC–MS/MS analysis, samples were desalted using an Empore C18 extraction column as per our earlier studies . Briefly, 30 µg of protein sample was employed in the C18 for each stage (200 μl pipette tips). A 40 μl of a 50% acetonitrile (ACN) solution was used to activate the desalting column. The liquid from the collection vial was centrifuged at 1000 g for 2 min at room temperature and discarded. The desalting column was cleaned with 40 μl of 99.9% ACN after the aforementioned step was repeated twice. Repeated centrifugation at 1000 g for 2 min at room temperature was used to remove the liquid from the collection vial. Formic acid (FA), solvent A, was used to equilibrate the desalting column twice using 40 μl of 0.1% (v/v) formic acid. Afterward, the centrifugation process was repeated and the liquid discarded. The samples were reconstituted in 40 μl of 0.1% (v/v) FA and vortexed slowly for 10 min. Reconstituted samples were spun at 1000 g for two minutes using a syringe or centrifuge to pass through the desalting column. This process was repeaed five times. A 50 μl sample of 40% (v/v) acetonitrile in 0.1% (v/v) FA was passed through the column and the elute was collected in a fresh 1.5 ml Eppendorf tube. The same vial was used to collect the elute after repeating the step with 50% and 60% (v/v) acetonitrile in 0.1% (v/v) FA. A 150 μl of the desalted sample was thereafter dried in a speed vacuum centrifuge. The desalted peptides were stored at -20 °C. Liquid chromatography-tandem mass spectrometry in data-dependent acquisition mode Peptides were measured for each sample (T1R1, T1R2, T1R3, T2R1, T2R2, T2R3, and T3R1, T3R2, T3R3) using the Scopes method for liquid chromatography-tandem mass spectrometric analysis (LC–MS/MS). On an Easy-nLC 1200 system, a 120-min gradient was applied to all samples. The peptide sample was placed into the trap column (100 mm × 2 cm, nanoViper C18, 5 mm, 100A; Thermo Fisher Scientific) at a flow rate of 5 μl/min. Peptides were separated on an analytical column (3 μm particle, and 100 Å pore size, 75 μm × 50 cm; Thermo Fisher Scientific) at a flow rate of 300 nl/min in solvent A (0.1% formic acid (FA) in water) and solvent B (80% acetonitrile with 0.1% FA) over a 120 min gradient). The mass spectrometry data were collected using an Orbitrap spectrometer connected to the aforementioned LC. The Orbitrap mass analyzer was used to perform mass spectrometric acquisition in data-dependent acquisition (DDA) mode over the entire scan range of 375–1700 m/z and with a mass resolution of 60,000. The mass window was set at 10 ppm with a dynamic exclusion time of 40 s. For fragmentation, all MS/MS spectra were obtained using the HCD method (High Energy Collision Dissociation). The AGC target for MS1 and MS2 was set at 400,000 and 10,000 respectively. A lock mass of 445.12003 m/z was used for positive internal calibration. MaxQuant analysis for proteome The raw mass spectrometry was analysed in MaxQuant (v1.6.6.0) software against the UniProt protein database for Labeo rohita using the in-built search engine, Andromeda. All raw files were analyzed together using Label-Free-Quantification (LFQ) parameters. Parameters include label type as standard with a multiplicity of 1 and the match between runs was checked. Orbitrap fusion mode was selected and trypsin as protease was used. A maximum of 2 missed cleavages were allowed. Under the fixed modification, carbamidomethylation at Cysteine (+ 57.021464 Da) and for variable modification, oxidation at Methionine (+ 15.994915 Da) was selected. Both at the protein and peptide levels, a false discovery level was kept at 1%. For the decoy mode option, the reverse was selected and proteins were identified with unique peptides only. For quantitative analysis, LFQ intensities obtained for each sample were selected. 

The confluent LR muscle cells at subcultures 1 (T1(R1, R2, R3)), 15 (T2 (R1, R2, R3)), and 25 (T3 (R1, R2, R3)) containing triplicate flasks for each replicates were harvested (2 × 10 6 ) using trypsin versene (Himedia, Mumbai) and the cells dislodged for 1 min. Harvested cells were homogenized in 500 µl of lysis buffer (7 M Urea, 2 M Thiourea, 4% CHAPS, 10 mM DTT, 5 µl protease inhibitor). Sonication was performed for 2 min with an amplitude of 40% and a 5-s pulse on and off. The tubes were centrifuged at 12,000 g for 10 min at 4 °C, and the supernatant was collected and incubated with 500 µL of 100% ice-chilled acetone (− 20 °C) overnight. Pellet formation was by centrifugation at 12,000 g for 10 min at 4 °C and the pellet was washed thrice with 80% chilled acetone. The resulting pellet was solubilized in the lysis buffer for 1–2 h in a vortex at room temperature. Centrifugation was performed for final protein extraction at 12,000 g for 10 min at 4 °C, and the supernatant was collected and stored at − 80 °C until further use. 

Cell lysates were processed for quantification of proteins using the Bradford method using bovine serum albumin as the standard. Briefly, five BSA standards were prepared at 10, 20, 30, 40, and 50 µg/ml and extracted samples were analyzed in a NanoDrop™ One/OneC Microvolume UV–Vis Spectrophotometer (Thermo Scientific). The in-solution-based digestion method was used to process 30 µg of protein. Prior to LC–MS/MS analysis, samples were desalted using an Empore C18 extraction column as per our earlier studies . Briefly, 30 µg of protein sample was employed in the C18 for each stage (200 μl pipette tips). A 40 μl of a 50% acetonitrile (ACN) solution was used to activate the desalting column. The liquid from the collection vial was centrifuged at 1000 g for 2 min at room temperature and discarded. The desalting column was cleaned with 40 μl of 99.9% ACN after the aforementioned step was repeated twice. Repeated centrifugation at 1000 g for 2 min at room temperature was used to remove the liquid from the collection vial. Formic acid (FA), solvent A, was used to equilibrate the desalting column twice using 40 μl of 0.1% (v/v) formic acid. Afterward, the centrifugation process was repeated and the liquid discarded. The samples were reconstituted in 40 μl of 0.1% (v/v) FA and vortexed slowly for 10 min. Reconstituted samples were spun at 1000 g for two minutes using a syringe or centrifuge to pass through the desalting column. This process was repeaed five times. A 50 μl sample of 40% (v/v) acetonitrile in 0.1% (v/v) FA was passed through the column and the elute was collected in a fresh 1.5 ml Eppendorf tube. The same vial was used to collect the elute after repeating the step with 50% and 60% (v/v) acetonitrile in 0.1% (v/v) FA. A 150 μl of the desalted sample was thereafter dried in a speed vacuum centrifuge. The desalted peptides were stored at -20 °C. 

Peptides were measured for each sample (T1R1, T1R2, T1R3, T2R1, T2R2, T2R3, and T3R1, T3R2, T3R3) using the Scopes method for liquid chromatography-tandem mass spectrometric analysis (LC–MS/MS). On an Easy-nLC 1200 system, a 120-min gradient was applied to all samples. The peptide sample was placed into the trap column (100 mm × 2 cm, nanoViper C18, 5 mm, 100A; Thermo Fisher Scientific) at a flow rate of 5 μl/min. Peptides were separated on an analytical column (3 μm particle, and 100 Å pore size, 75 μm × 50 cm; Thermo Fisher Scientific) at a flow rate of 300 nl/min in solvent A (0.1% formic acid (FA) in water) and solvent B (80% acetonitrile with 0.1% FA) over a 120 min gradient). The mass spectrometry data were collected using an Orbitrap spectrometer connected to the aforementioned LC. The Orbitrap mass analyzer was used to perform mass spectrometric acquisition in data-dependent acquisition (DDA) mode over the entire scan range of 375–1700 m/z and with a mass resolution of 60,000. The mass window was set at 10 ppm with a dynamic exclusion time of 40 s. For fragmentation, all MS/MS spectra were obtained using the HCD method (High Energy Collision Dissociation). The AGC target for MS1 and MS2 was set at 400,000 and 10,000 respectively. A lock mass of 445.12003 m/z was used for positive internal calibration. 

The raw mass spectrometry was analysed in MaxQuant (v1.6.6.0) software against the UniProt protein database for Labeo rohita using the in-built search engine, Andromeda. All raw files were analyzed together using Label-Free-Quantification (LFQ) parameters. Parameters include label type as standard with a multiplicity of 1 and the match between runs was checked. Orbitrap fusion mode was selected and trypsin as protease was used. A maximum of 2 missed cleavages were allowed. Under the fixed modification, carbamidomethylation at Cysteine (+ 57.021464 Da) and for variable modification, oxidation at Methionine (+ 15.994915 Da) was selected. Both at the protein and peptide levels, a false discovery level was kept at 1%. For the decoy mode option, the reverse was selected and proteins were identified with unique peptides only. For quantitative analysis, LFQ intensities obtained for each sample were selected. 

Statistical analysis was performed in metaboanalyst across different passages of LR muscle cells T1 vs T2, T2 vs T3, and T1 vs T3 by taking MaxQuant analyzed files . The proteomic data of the nine samples were considered for further analysis. Missing values of the features having abundance values of more than 75% of each group were imputed by the KNN algorithm and considered for differential protein expression analysis. Log-transformed data along with normalization was taken for statistical analysis (i.e., t-test and fold change). Two sample t-test (Welch t-test) was performed to identify the significant differentially expressed proteins where a p value of 0.05 was set as the maximum threshold value. Among all t-test passed proteins, proteins with a fold change value of 1.5 were considered significantly differentially expressed proteins (DEPs). 

LFQ data of different passages were taken forward for functional annotation and biological pathway analysis. The gene ontology classification of dysregulated proteins was performed in the eggNOG (Version 6.0) . Later, the entire identified abundant proteome gene list was analyzed using Metascape between the passages . In addition, the network visualization and the protein–protein interaction analysis for selected pathways were done in STRING between the passages (version 11.0) . 

Labeo rohita muscle cell culture was successfully developed and characterized using proteome analysis. Comparative proteome analysis of LR muscle cells revealed a total of 138 proteins containing at least two unique peptides. In total, 120 unique proteins were differential expressed in all the conditions. The enriched pathways underscore the critical roles of glycolytic (eno1 and eno3), metabolic (smyd1, ak1, aldob, and bpgm), cytoplasmic ribosomal (rpl27, rpl3, and eif5A) and carbon metabolic (pdh) during muscle cell growth and differentiation across passages 1, 15 and 25. The proteins observed across the pathways such as annexins, ribosomal proteins, and metabolic enzymes revealed dynamic changes that align with the transition from proliferative to differentiated states of LR muscle cells during myogenesis. Moreover, the developed muscle cells would be useful for understanding in vitro myogenesis and other aspects of cultivated seafood development. Obatined proteins were crucial for cell proliferation and differentiation for developing optimized cultured meat production. 

Supplementary Information. 

