The muscular system is an important organ system responsible for the body movement and is composed of different protein components [1]. In addition to the body movement, muscular system has other functions including the maintenance of body posture and position, movement of substances inside the body and generation of body heat.
The interactions between the proteins that are found in the muscle fibers drive the body movements from swimming to breathing. Muscle cells contain myofibrils which are the basic contractile elements, and those are bundled into muscle fibers [4]. Sarcomeres, which are the linear contractile units are found in each myofibril and are the assemblies of actin(thin) and myosin(thick) protein filaments aligned in a parallel manner [4]. ATP is hydrolyzed by myosin which causes the shortening of sarcomere and myosin moves along the actin filaments, this in turn results in, sarcomere ends to be pulled towards each other [7]. Many sarcomeres along the muscle fiber contract in this fashion leading to entire muscle to contract [7].
Myosin and actin proteins are conserved across different species [1]. However, the molecular weight of myosin light chain varies across different species depending on the environment the species live in, the food they eat and different physiological stresses they face [1]. Thus, by studying the molecular weight of myosin light chain with the use of Western blots, these variations between different species can be analyzed.
Other than conserved actin and myosin proteins, there are various other proteins that aid in contraction of muscles which show more variability from one species to another. Therefore, these other proteins that show variability can be studied to reflect the modifications to muscle performance and function which are adaptive to different environments, and niches [8]. Furthermore, they can be used to study how closely the species are related to one another [8].
Proteome is the collection of all proteins of a cell, tissue or organism and differ from cell to cell, tissue to tissue and organism to organism [1] Thus, proteomics is the study of structure and functions of all proteins. Comparative proteomics aims to analyze the differences between the proteome of organisms in response to development, environment, and diseases [2]. Physiological adaptations to diverse niches and environment are reflected in organisms’ variations in proteins which result from mutations in DNA. The mutations that are favorable persist through natural selection resulting in evolution of new species with new specialized functions [1]. Therefore, different proteins that are found in different species can be an indicative of genetic and evolutionary relatedness across species [2].
One of the purposes of this experiment was to analyze the differences in muscle proteins that can be indicative of the evolutionary relatedness of three different fish species (Tilapia, Shark, and Cod), by using comparative proteomics and the molecular biology methods such as extraction, SDS- PAGE, and Coomassie staining of proteins.
The second purpose of this experiment was to study the differences in molecular weights of myosin light chain to analyze how the environment, niches and physiological stress vary across three different fish species, through the use of Western blotting method.
Material and Methods:
First of all, fish tissues from different fish species (Cod, Shark, or Tilapia) were obtained. Protein extraction was performed by adding 250 L Laemmli (1xSDS) sample buffer and minced fish tissues into1.5 ml microcentrifuge tube. The tube was gently flicked to agitate the tissue and was incubated at room temperature for 5 minutes. Tube was then shaken down to pellet the tissues, and the supernatant was transferred to new 1.5 mL screw cap tube. The sample was then boiled for 5 minutes at 95 oC.
Two precast SDS-PAGE TGX gels (one for Coomassie Blue staining and other for immunoblotting) from Bio-Rad were used to run the samples. The gel apparatus used was MINI-PROTEAN Gels in Mini-PROTEAN Tetra Cell. The comb was removed from the top and the tape was peeled off from the bottom of the cassettes. Gel cassettes were assembled and placed into the tank. Buffer chambers were then filled up to the ‘2 Gel’ line with 1x running buffer. Sample wells were all washed with running buffer. 10 L (Coomassie staining) and 5 l(immunoblotting) of each sample was added into the gels. For each gel, 7 L of protein ladder and 5 L of Actin/Myosin Standards were added to lane #1 and #6, respectively. The gels ran at 200V for 30-40 minutes and was stopped when the dye reached the bottom of the cassettes. The cassettes were disassembled from the tank and two cassette plates were pulled apart. The gel for Coomaasie staining was placed into a container with deionized water and incubated on a rocking platform for 5 minutes. The gel was then transferred into Coomassie staining solution and was rocked for 1 hour. Afterwards, the gel was placed back into deionized water and incubated for at least 15 minutes on a rocking platform.
Transfer of immunoblot gels onto the membrane (PVDF) was done using BioRad Transfer packs. The bottom stack, membrane, the gel and the second wetted transfer stack were placed in order (down to up) on the cassette base. Cassette was placed into turbo blotter to transfer the gel onto the membrane. For immunoblotting, a primary blocking step was performed in a blocking solution (5% non-fat milk in 1xTBS-T) for at least 1 hour. 5L of the primary antibody (Mouse Anti-Myosin Light Chain Antibody) was then added into the blocking solution and the plate was incubated for 20 mins on the rocker. After, solution was drained and 15mL Wash Buffer (1xTBS-T) was added to plates and incubated at rocker for at least 10 minutes. This procedure was repeated for 2 additional times. After the 3rd wash, 15 mL blocking solution and 5L of the secondary antibody were added (Goat Anti-Mouse Antibody). Plate was swirled for 5 seconds and incubated for 15 minutes on rocker. 3 more washes were done each with 15 mL wash buffer for 10 minutes. Then the Reagents (A&B) of substrate kit components were mixed by adding 200L of each. Substrate was spread evenly across the middle of the blot and incubated for 5 minutes. Digital imager Bio-Rad ChemiDoc MP Imaging System was used to view the bands on the membrane. For molecular weight and relative intensity analysis of bands on both gels, ImageLab software (Version 6.0) was used.
Materials and Methods was adapted from BIO314 Laboratory Manual [1].
Results:
Although there is some smearing at the top of the gel, overall the quality of the gel from Coomassie staining shown in Figure 1, is good, and can be used for comparative proteomics. Also, lanes 5,7,8, 9, and 10 showed indistinct bands around 30-40 kDa. Not all of the bands were present for all of the species and the intensities of the bands differed from one species to another. E.g., the intensity of bands in lanes 4 and 5 were lower than those bands in lanes 7 and 8. Some of the bands that are present are likely to correspond to known muscle proteins, for instance in all lanes there were bands of ~ 30kDa, which corresponds to troponin. In myosin light chain region, intensities of the bands for Cod and Shark were higher than that of Tilapia, however the molecular weights(MW) were almost the same across the 3 species. The differences, between the intensities and MWs of the other proteins for all fish species could be indicative that they are not very closely related.
In figure 2, from immunoblotting, there was only 1 band from lane 1 to 8, however the bands were absent in the lanes 9 and 10. Size of the bands for the lanes 1 to 8 were between 21-23 kDa, with the smallest one being 21.9 belonging to Tilapia, and the highest one being 23.1 belonging to Shark. As shown in table 1, Shark had a MW closest to the MW of the marker. Tilapia had a lower MW compared to the marker. Cod showed less MW in lanes 2 and 4 when compared to the marker, however in lane 8 MW of Cod was really close to the marker. The intensities of the bands for both Shark and Cod were much higher when compared with marker, however Tilapia had a lower intensity than the marker.
The blot results showed that MWs of the myosin light chain of Shark and Cod were close to each other and to the markers, the Coomasie result on the other hand indicated all 3 species had around the same MW in the myosin light chain region. Furthermore, the intensities of the bands for Cod and Shark were higher compared to markers and Tilapia in both results, indicating overall higher protein mass. These results could be showing that Cod and Shark have the same overall myosin light chain mass and could be indicative that they live in similar environments and face the same physiological stress. However, because the gel only had 1 tilapia sample, these results could be biased.
Discussion:
The results of these experiments showed that Cod, Tilapia and Shark had different molecular weights(MW) and intensities for the muscle proteins. Coomassie stained gel showed that, around the myosin light chain(MLC) region all the three fish had approximately the same MW, this was not consistent with the results from the immunoblot gel, where Cod and Shark had approximately the same MW while the Tilapia had lower. However, both results from Coomassie and immunoblot gels showed that Cod and Shark had a higher intensity for the MLC protein, where for Tilapia intensity was lower.
One of the limitations with the experiment was that both gels had Tilapia sample only once, which could have been biasing these findings. Another experiment can be done using the equal number of the samples from all three fish species to eliminate this bias. Another limitation was that last two lanes in the immunoblot gel was missing, this could be due to having much lower extract concentration, or simply samples were not loaded into the wells. This issue could be eliminated by measuring the extract concentration using spectrophotometric methods to ensure the comparison of the proteins are on equal basis [3].
In this experiment, MLC was specifically immuno-probed by the primary antibody Mouse anti-myosin and the secondary antibody (Goat anti-mouse HRP) was used for binding to the primary antibody. This immuno-probing showed that for all of the three fish species MLC was present, but seemed to be less intense in Tilapia. This indicated that both Shark and Cod had higher overall mass of myosin in their muscle composition. MW of the myosin was also high in both Cod and Shark and was low in Tilapia. These could be reflecting that the Cod and Shark face the similar environmental conditions.
Immunoblotting gave more specific results about the MLC, which generated the visualization of only the MLC protein in a sample. This was extremely useful in comparing why these three fish species have varying mass of myosin in their muscles, which was a highly conserved protein through all of these species. This gave insights into the how the function of the muscle was altered through the change in the protein mass due to the different environmental conditions, life styles, and physiological stresses. However, to draw firmer conclusions, it might be better to compare the same fish species under varying environmental conditions and study the MW and the intensities of the MLC using Western blotting. The results from this type of experiment could be used to strongly conclude the changes in the mass of the MLC protein is only due to the different environmental conditions and niches.
Both conserved proteins myosin and actin were present in all samples. However, the samples showed many differences in other proteins that compose the muscle tissue. These differences could be indicative that the genome of the three species are not similar and they are not related to one another through the same common ancestor. Muscles of different species needs to be adaptive with new specialized functions to the changing environmental conditions and stresses. Therefore, the differences in the muscle proteome could arise due to the mutation in the genome of an organism, selected through the natural selection, which results in useful physiological adaptations to the diverse environments and niches.
Proteins are the products of central dogma and can be used as species ID tool. Thus, the amount and type of proteins from different organisms can be compared to one another to get an insight into how closely related different species are or how different species’ genomes are similar to one another [5]. Furthermore, proteins can also be used as biological markers that could aid in diagnosing a specific disease [6]. Proteomes of patients with disease can be screened for specific amount of protein that is found in specific disease state [6].
In conclusion, findings of these experiments showed that myosin and actin was conserved through different species, with other proteins varying from species to species. MW and intensities of these other variable proteins were different across the fish species. Specifically, the MW and intensity of MLC protein was approximately the same for Cod and Shark and were lower for that of Tilapia. These findings were valuable for the purposes of this experiment, where they could be indicative that these fish were not related to one another through a common ancestor. However, Cod and Shark might be living under similar environmental conditions and facing similar physiological stresses.