Application of mass spectrometry-based proteomics to muscle biology and meat science
Date
2024-05
Authors
Johnson, Logan Gregory
Major Professor
Advisor
Lonergan, Steven M
Huff-Lonergan, Elisabeth J
Nair, Mahesh N
Steibel, Juan P
Liu, Peng
Committee Member
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Abstract
Livestock, meat, and food production systems have improved and become more efficient in the last few decades. The efficiency gains are partly due to the fundamental knowledge of the impacts of genetic selection, nutritional strategies, health interventions, facilities and welfare, and other technological innovations on the growth and efficiency of livestock. The eating quality of meat products remains challenging to predict, and undesirable meat products contribute to the overall waste of the production systems. Improving meat product quality could help reduce food product waste, improve access to meat products by a growing global population, and increase the value of meat products. Fundamental knowledge of factors impacting and relating to meat quality must be developed to balance productivity gains with value-related economic factors. A large body of research has documented that changes in the molecular features of postmortem muscle, including pH decline, lipid and protein oxidation, and protein degradation, result in measurable impacts on meat quality. Specifically, the degradation of structural proteins in postmortem muscle improves tenderness. Calpain proteinases contribute to postmortem proteolysis and degradation of specific cytoskeletal and myofibrillar proteins, which are related to improved meat quality and tenderness. Recognition of the molecular factors that can affect calpain activity and the susceptibility of proteins to degradation by calpains will generate robust information that can extend the knowledge of postmortem muscle biochemistry. In characterizing better these molecular factors, more robust and reliable tools to genetically select for meat quality and to categorize meat products based on their quality.
The application and utility of mass spectrometry-based methods can allow for discovering and characterizing proteomic features associated with postmortem meat quality in a non-targeted or shot-gun approach. These mass spectrometry platforms can also be applied to hypothesis-based research questions. Both approaches have and will continue to provide crucial molecular information to improve the efficient production of livestock and meat products. This dissertation will explore both strategies of broader, discovery-based, and more targeted approaches to provide a greater understanding of proteome and protein differences related to postmortem muscle and meat quality. Therefore, the specific objectives of this dissertation were to 1) Identify the sarcoplasmic sub-proteomic differences between groups of aged pork chops based on water-holding capacity, 2) Determine the myofibrillar sub-proteomic differences between groups of pork chops based on instrumental tenderness values, 3) Understand the impacts of secondary lipid oxidation products on calpain-2 activity and autolysis and determine the quantity and localization of modification sites, and 4) Train machine learning models with specific tryptic peptide abundances to predict the ~2-week aged pork chop instrumental tenderness value.
Lipid and protein oxidation occurs in postmortem skeletal muscle, and the products of these reactions generally accumulate during the postmortem period. The oxidation products of lipids can be aldehyde, ketone, and other reactive products, which have been shown to modify and impact proteins’ functionality. Previous work has demonstrated that some lipid oxidation products can covalently modify calpain-1, resulting in activity and autolysis differences. The current work presented similar observations and showed that calpain-2 activity and the progression of autolysis were impacted differently by malondialdehyde, 2-hexenal, and 4-hydroxynonenal. Specific modification sites were determined with LC-MS/MS, including distinct malondialdehyde and 2-hexenal modification sites. Intact protein mass analysis with MALDI-MS revealed that many modifications on calpain-2 catalytic and regulatory subunits are likely to exist. These modifications can potentially occur in living and postmortem skeletal muscle, impact the solubility, functionality, and cellular localization of calpain-2 and other proteins, and alter the development of meat quality.
Proteomic analysis using liquid chromatography-mass spectrometry (LC-MS) and tandem mass tags (TMT) has only recently been applied to research questions related to meat science. Herein, the utility of the fractionation of proteins based on solubility in a low-ionic strength buffer to reveal specific molecular differences related to pork quality is demonstrated. Indeed, this approach revealed potential proteolytic degradation products, including titin, desmin, and filamin-C, accumulating in the sarcoplasmic sub-proteome and more abundant in pork chop groups with less purge loss. Proteins related to the sarcoplasmic reticulum, calcium-regulating, and chaperone proteins, including calsequestrin, calcium transport ATPases, and heat shock proteins, were generally more abundant in these samples with less purge loss. In another experiment, the aged myofibrillar sub-proteome was fractionated to evaluate specific proteomic differences related to the instrumental star probe of pork chops. A lower abundance of proteins related to the Z-line and metabolic-associated proteins, such as titin, desmin, nebulin, PDZ and LIM domain type proteins, and glycogen phosphorylase, was observed in the myofibrillar sub-proteome of more tender pork chops. These observations help refine the knowledge of proteomic differences and changes related to pork quality.
Proteomics experiments utilizing bottom-up approaches commonly result in an emphasis on protein abundance differences. In fact, two experiments in this dissertation have reported on differences in protein abundance. However, postmortem muscle's molecular changes and phenotype result in a more complex proteomic profile. The modifications of proteins, including conjugation, denaturation, oxidation, and degradation, generate a different profile of proteins, including intact and degraded forms of many proteins. The recognition of the limitations of current bottom-up approaches to identify degradation products led to the idea that the abundance of tryptic peptides generated from proteins soluble in a low-ionic strength buffer may be more informative and helpful in predicting the ultimate meat quality. Using feature-selected peptides, different machine learning models were trained and assessed to predict the ultimate instrumental star probe value. Machine learning models trained using only physical quality measures resulted in models with low (< 0.2) R2 values. The feature-selected peptides produced better fitting models, with higher (~ 0.5) R2 and lower root mean squared error and mean absolute error when predicting the continuous instrumental star probe value. The feature-selected peptides important in the machine learning models were identified to be primarily structural proteins, which are likely products of proteolysis in the aged sarcoplasmic sub-proteome. These data demonstrate the utility of specific tryptic peptides to predict pork tenderness.
This research demonstrates the importance of understanding factors that influence and are related to meat quality differences. These molecular features are relevant and important in better characterizing the changes occurring in postmortem skeletal muscle. This work investigated proteomic differences in different protein fractions, factors that may impact calpain-2 functionality, and the utility of specific peptide features to improve the prediction of meat tenderness. Specific post-translational modifications and the solubility changes of protein and peptide degradation products are essential aspects less appreciated in the literature and warrant greater investigation, characterization, and validation. Notably, the work presented herein also provides a framework for future work that can refine understanding by applying fractionation of samples before bottom-up techniques. Top-down mass spectrometry approaches will become more commonly utilized techniques with improved instrumentation, and the work with these approaches lays the groundwork for subsequent research investigations.
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dissertation