Secondary Lipid Oxidation Products as Modulators of Calpain-2 Functionality In Vitro.

The objective was to understand the impacts of secondary lipid oxidation products on calpain-2 activity and autolysis and, subsequently, to determine the quantity and localization of modification sites. 2-Hexenal and 4-hydroxynonenal incubation significantly decreased calpain-2 activity and slowed the progression of autolysis, while malondialdehyde had minimal impact on calpain-2 activity and autolysis. Specific modification sites were determined with LC-MS/MS, including distinct malondialdehyde modification sites on the calpain-2 catalytic and regulatory subunits. 2-Hexenal modification sites were observed on the calpain-2 catalytic subunit. Intact protein mass analysis with MALDI-MS revealed that a significant number of modifications on the calpain-2 catalytic and regulatory subunits are likely to exist. These observations confirm that specific lipid oxidation products modify calpain-2 and may affect the calpain-2 functionality. The results of these novel experiments have implications for healthy tissue metabolism, skeletal muscle growth, and post-mortem meat tenderness development.

Distinct myofibrillar sub-proteomic profiles are associated with the instrumental texture of aged pork loin.

Fresh pork tenderness contributes to consumer satisfaction with the eating experience. Postmortem proteolysis of proteins within and between myofibrils has been closely linked with pork tenderness development. A clear understanding of the molecular features associated with pork tenderness development will provide additional targets and open the door to new solutions to improve and make pork tenderness development more consistent. Therefore, the objective was to utilize liquid chromatography and mass spectrometry with tandem mass tag (TMT) multiplexing to evaluate myofibrillar sub-proteome differences between pork chops of different instrumental star probe values. Pork loins (N = 120) were collected from a commercial harvest facility at 24 h postmortem. Quality and sensory attributes were evaluated at 24 h postmortem and after ~2 weeks of postmortem aging. Pork chops were grouped into 4 groups based on instrumental star probe value (group A,x¯â€…= 4.23 kg, 3.43 to 4.55 kg; group B,x¯â€…= 4.79 kg, 4.66 to 5.00 kg; group C,x¯â€…= 5.43 kg, 5.20 to 5.64 kg; group D,x¯â€…= 6.21 kg, 5.70 to 7.41 kg; n = 25 per group). Myofibrillar proteins from the samples aged ~2 wk were fractionated, washed, and solubilized in 8.3 M urea, 2 M thiourea, and 1% dithiothreitol. Proteins were digested with trypsin, labeled with 11-plex isobaric TMT reagents, and identified and quantified using a Q-Exactive Mass Spectrometer. Between groups A and D, 54 protein groups were differentially abundant (adjusted P < 0.05). Group A had a greater abundance of proteins related to the thick and thin filament and a lesser abundance of Z-line-associated proteins and metabolic enzymes than group D chops. These data highlight that distinct myofibrillar sub-proteomes are associated with pork chops of different tenderness values. Future research should evaluate changes immediately and earlier postmortem to further elucidate myofibrillar sub-proteome differences over the postmortem aging period.

A primary goal of meat production is to efficiently produce safe, high-quality products. Competing interests within the goal complicate this seemingly simple aspiration. Consequently, it is necessary to emphasize efforts to enhance our comprehension of biological and molecular factors that influence quality, safety, and efficient meat production. This experiment aimed to define the proteomic profiles of the myofibrillar fraction of fresh pork with differing quality traits. Myofibrils from aged pork chops with a range of tenderness levels were used to achieve this objective. Fifty-four proteins were differentially abundant between the divergent tenderness groups. This was due to the expression profile of proteins in muscle and/or changes in proteins in the myofibrillar fraction during postmortem aging. These results inform and direct the development of antemortem and postmortem applications to ensure success in producing high-quality pork.

Lipid Peroxidation Products Influence Calpain-1 Functionality In Vitro by Covalent Binding.

The objective of the current study was to evaluate the effects of lipid peroxidation products, malondialdehyde (MDA), hexenal, and 4-hydroxynonenal (HNE), on calpain-1 function, and liquid chromatography and tandem mass spectrometry (LC-MS/MS) identification of adducts on calpain-1. Calpain-1 activity slightly increased after incubation with 100 µM MDA but not with 500 and 1000 µM MDA. However, calpain-1 activity was lowered by hexenal and HNE at 100, 500, and 1000 µM. No difference in calpain-1 autolysis was observed between the control and 1000 µM MDA. However, 1000 µM hexenal and HNE treatments slowed the calpain-1 autolysis. Adducts of MDA were detected on glutamine, arginine, lysine, histidine, and asparagine residues via Schiff base formation, while HNE adducts were detected on histidine, lysine, glutamine, and asparagine residues via Michael addition. These results are the first to demonstrate that lipid peroxidation products can impact calpain-1 activity in a concentration-dependent manner and may impact the development of meat tenderness postmortem.

Characterizing the sarcoplasmic proteome of aged pork chops classified by purge loss.

Unpredictable variation in quality, including fresh pork water-holding capacity, remains challenging to pork processors and customers. Defining the diverse factors that influence fresh pork water-holding capacity is necessary to make progress in refining pork quality prediction methods. The objective was to utilize liquid chromatography and mass spectrometry coupled with tandem mass tag (TMT) multiplexing to evaluate the sarcoplasmic proteome of aged pork loins classified by purge loss. Fresh commercial pork loins were collected, aged 12 or 14 d postmortem, and pork quality and sensory attributes were evaluated. Chops were classified into Low (N = 27, average purge = 0.33%), Intermediate (N = 27, average purge = 0.72%), or High (N = 27, average purge = 1.19%) chop purge groups. Proteins soluble in a low-ionic strength buffer were extracted, digested with trypsin, labeled with 11-plex isobaric TMT reagents, and detected using a Q-Exactive Mass Spectrometer. Between the Low and High purge groups, 40 proteins were differentially (P < 0.05) abundant. The Low purge group had a greater abundance of proteins classified as structural and contractile, sarcoplasmic reticulum and calcium regulating, chaperone, and citric acid cycle enzymes than the High purge group. The presence of myofibrillar proteins in the aged sarcoplasmic proteome is likely due to postmortem degradation. These observations support our hypothesis that pork chops with low purge have a greater abundance of structural proteins in the soluble protein fraction. Together, these and other proteins in the aged sarcoplasmic proteome may be biomarkers of pork water-holding capacity. Additional research should establish the utility of these proteins as biomarkers early postmortem and over subsequent aging periods.

Fresh pork can vary in its ability to retain water­commonly termed as its water-holding capacity­where a greater water-holding capacity means it retains more water as it is cut, packaged, and stored. However, commercial pork loins have considerable variability in their water-holding capacity, which can impact the consumer's eating experience. This study aimed to examine water-soluble proteins from aged commercial pork chops and to identify and quantify these proteins with mass spectrometry to confirm the previous observation that the degradation of specific structural proteins is associated with greater water-holding capacity. This analysis identified 40 proteins differentially abundant between pork chops with varying water-holding capacities. Pork chops with greater water-holding capacity had a greater abundance of proteins classified as structural and contractile, calcium regulating, and chaperone. Metabolic proteins were also differentially abundant in aged pork loins with differing water-holding capacity. This study confirmed previous observations that the degradation of key structural proteins is associated with greater water-holding capacity while identifying new proteins that may be biomarkers for water-holding capacity.

Evaluating the ability of rapid evaporative ionization mass spectrometry to differentiate beef palatability based on consumer preference.

Rapid Evaporative Ionization Mass Spectrometry (REIMS) is a type of ambient ionization mass spectrometry, which enables real-time evaluation of several complex traits from a single measurement. The objective of this study was to evaluate the capability of REIMS analysis of raw samples coupled with chemometrics to accurately identify and predict cooked beef palatability. REIMS analysis and consumer sensory evaluation were conducted for beef arm center roasts (n = 20), top loin steaks (n = 20), top sirloin steaks (n = 20), and 20% lipid ground beef (n = 20). These data were used to train predictive models for six classification sets representing different sensory traits. The maximum prediction accuracies achieved (from high to low): beefy flavor acceptance (86.25%), juiciness acceptance (83.75%), overall acceptance (81.25%), overall flavor acceptance (81.25%), grilled flavor acceptance (78.75%), and tenderness acceptance (75%). The current study demonstrates that REIMS analysis of raw meat has the potential to predict and classify cooked beef palatability. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05562-6.

Tandem mass tag labeling to assess proteome differences between intermediate and very tender beef steaks.

Tenderness is considered as one of the most important quality attributes dictating consumers' overall satisfaction and future purchasing decisions of fresh beef. However, the ability to predict and manage tenderness has proven very challenging due to the numerous factors that contribute to variation in end-product tenderness. Proteomic profiling allows for global examination of differentially abundant proteins in the meat and can provide new insight into biological mechanisms related to meat tenderness. Hence, the objective of this study was to examine proteomic profiles of beef longissimus lumborum (LL) steaks varying in tenderness, with the intention to identify potential biomarkers related to tenderness. For this purpose, beef LL muscle samples were collected from 99 carcasses at 0 and 384 h postmortem. Based on Warner-Bratzler shear force values at 384 h, 16 samples with the highest (intermediate tender, IT) and lowest (very tender, VT) values were selected to be used for proteomic analysis in this study (n = 8 per category). Using tandem mass tag-based proteomics, a total of 876 proteins were identified, of which 51 proteins were differentially abundant (P < 0.05) between the tenderness categories and aging periods. The differentially identified proteins encompassed a wide array of biological processes related to muscle contraction, calcium signaling, metabolism, extracellular matrix organization, chaperone, and apoptosis. A greater (P < 0.05) relative abundance of proteins associated with carbohydrate metabolism and apoptosis, and a lower (P < 0.05) relative abundance of proteins involved in muscle contraction was observed in the VT steaks after aging compared with the IT steaks, suggesting that more proteolysis occurred in the VT steaks. This may be explained by the greater (P < 0.05) abundance of chaperonin and calcium-binding proteins in the IT steaks, which could have limited the extent of postmortem proteolysis in these steaks. In addition, a greater (P < 0.05) abundance of connective tissue proteins was also observed in the IT steaks, which likely contributed to the difference in tenderness due to added background toughness. The established proteomic database obtained in this study may provide a reference for future research regarding potential protein biomarkers that are associated with meat tenderness.

Among all the eating quality attributes of beef, tenderness is considered an essential factor influencing consumers' overall satisfaction and future purchasing decisions. However, managing and predicting tenderness of meat products is challenging for the meat industry, as many factors can influence this attribute. The goal of this research was to examine variations in protein abundance between two categories of beef strip steaks varying in tenderness, with the intention to identify proteins related to beef tenderness/toughness. Overall, the results from this study suggest that tender steaks experienced greater protein degradation during aging than tougher steaks, which likely contributed to their improved tenderness. Furthermore, a greater abundance of connective tissue proteins, which are associated with meat toughness, was observed in the tougher steaks. Our results collectively indicate that the difference in tenderness between the two groups of steaks may be due to multiple proteins involved in several biological processes.

Pulmonary arterial pressure in fattened Angus steers at moderate altitude influences early postmortem mitochondria functionality and meat color during retail display.

Pulmonary hypertension is a noninfectious disease of cattle at altitudes > 1524 m (5,000 ft). Mean pulmonary arterial pressures (PAP) are used as an indicator for pulmonary hypertension in cattle. High PAP cattle (≥50 mmHg) entering the feedlot at moderate elevations have lower feed efficiency as compared to low PAP cattle (< 50 mmHg). The impact of pulmonary arterial pressure on mitochondrial function, oxidative phosphorylation (OXPHOS) protein abundance, and meat color was examined using longissimus lumborum (LL) from high (98 ± 13 mmHg; n = 5) and low (41 ± 3 mmHg; n = 6) PAP fattened Angus steers (live weight of 588 ± 38 kg) during early postmortem period (2 and 48 h) and retail display (days 1 to 9), respectively. High PAP muscle had greater (P = 0.013) OXPHOS-linked respiration and proton leak-associated respiration than low PAP muscles at 2 h postmortem but rapidly declined to be similar (P = 0.145) to low PAP muscle by 48 h postmortem. OXPHOS protein expression was higher (P = 0.045) in low PAP than high PAP muscle. During retail display, redness, chroma, hue, ratio of reflectance at 630 and 580 nm, and metmyoglobin reducing activity decreased faster (P < 0.05) in high PAP steaks than low PAP. Lipid oxidation significantly increased (P < 0.05) in high PAP steaks but not (P > 0.05) in low PAP. The results indicated that high PAP caused a lower OXPHOS efficiency and greater fuel oxidation rates under conditions of low ATP demand in premortem beef LL muscle; this could explain the lower feed efficiency in high PAP feedlot cattle compared to low PAP counterparts. Mitochondrial integral function (membrane integrity or/and protein function) declined faster in high PAP than low PAP muscle at early postmortem. LL steaks from high PAP animals had lower color stability than those from the low PAP animals during simulated retail display, which could be partially attributed to the loss of muscle mitochondrial function at early postmortem by ROS damage in high PAP muscle.

The impact of pulmonary arterial pressure (PAP) on mitochondrial function, oxidative phosphorylation protein abundance, and meat color was examined using longissimus lumborum (LL) from high (98 ± 13 mmHg) and low (41 ± 3 mmHg) PAP fattened Angus steers (live weight of 588 ± 38 kg) during early postmortem period (2 and 48 h) and retail display (days 1 to 9), respectively. The results indicated that high PAP caused a lower oxidative phosphorylation efficiency and greater fuel oxidation rates under conditions of positive energy balance in beef LL muscle. This could explain the lower feed efficiency in high PAP feedlot cattle compared to low PAP counterparts. Mitochondrial integral function declined faster in high PAP than low PAP muscle at early postmortem. LL steaks from high PAP animals had lower color stability than those from the low PAP animals during simulated retail display, which could be partially attributed to the loss of muscle mitochondrial function at early postmortem in high PAP muscle.

An early-postmortem metabolic comparison among three extreme acute heat stress temperature settings in chicken breast muscle.

Normally, preslaughter acute heat stress could accelerate postmortem glycolysis and impair chicken breast (pectoralis major muscle) quality. However, previous studies indicated that it might be different when the acute heat stress temperature rises to an extreme range (above 35 °C). Therefore, this study's objectives were to compare the pH decline, glycolytic enzyme activity, and AMP-activated protein kinase (AMPK) phosphorylation at early postmortem among three extreme acute heat stress temperature settings: a control group (36 °C) and two experimental groups (38 °C and 40 °C). Although the temperature did not affect glycogen phosphorylase a and pyruvate kinase activity, there was a decrease in pH decline rate, phosphofructokinase-1 activity, and phospho-AMPK-α[Thr172] within 4 h postmortem when temperature increased from 36 to 40 °C. Temperature also affected hexokinase activity, with the 36 °C-group having the highest activity. The results of the current study, for the first time, indicated that postmortem metabolic rate in chicken breast muscle could be changed by acute heat stress temperature setting at extreme range.

Tandem mass tagged dataset used to characterize muscle-specific proteome changes in beef during early postmortem period.

Bovine longissimus lumborum (LL) and psoas major (PM) muscles biopsy samples were collected from four carcasses (n = 4) at 45 min, 12 h, and 36 h postmortem from a commercial beef processing facility. Proteins present in the early postmortem LL and PM proteomes were identified and quantified using tandem mass tag (TMT) labelled, fractionated peptides coupled to liquid chromatography tandem mass spectrometry (LC-MS/MS). The data are supplied in this article and are related to "Tandem mass tag labeling to characterize muscle-specific proteome changes in beef during early postmortem period" by Zhai et al. [1].

Tandem mass tag labeling to characterize muscle-specific proteome changes in beef during early postmortem period.

Previous research has indicated that variation in the proteome profile of longissimus lumborum (LL) and psoas major (PM) post-rigor influences meat quality attributes such as tenderness and color stability during retail display. Tandem mass tag (TMT) labeling is a chemical labeling approach using isobaric mass tags for accurate mass spectrometry-based quantification and identification of biological macromolecules. The objective of this study was to use TMT labeling to examine proteome profile variation between beef LL and PM during the early postmortem period (45 min, 12 h, and 36 h). We identified a total of 629 proteins, of which 71 were differentially abundant (fold change > 1.5, P < .05) from three comparisons between the muscles (PM vs. LL at 45 min, 12 h and 36 h). These proteins were mainly involved in oxidative phosphorylation and ATP-related transport, tricarboxylic acid cycle, NADPH regeneration, fatty acid degradation, muscle contraction, calcium signaling, chaperone activity, oxygen transport, as well as degradation of the extracellular matrix. At early postmortem, more abundant antiapoptotic proteins in LL could cause high metabolic stability, enhanced autophagy, and delayed apoptosis, while overabundant metabolic enzymes and pro-apoptotic proteins in PM could accelerate the generation of reactive oxygen species and initiation of cell death. SIGNIFICANCE: Longissimus lumborum and psoas major are two highly valuable cuts in beef carcass exhibiting different quality attributes such as color and tenderness, partly due to their distinct muscle fiber proportion and metabolic property. We characterized the proteomic change of these two muscles at early postmortem using tandem mass tag labeling for the first time, which highlighted the potential relationships between metabolism, cell death, and color stability. Our work provides a new perspective on muscle-to-meat conversion, especially on the potential effect of muscle-specific cell death on meat color stability.

Carbon Chain Length of Lipid Oxidation Products Influence Lactate Dehydrogenase and NADH-Dependent Metmyoglobin Reductase Activity.

The biochemical basis of lower metmyoglobin reducing activity (MRA) in high-oxygen modified atmospheric packaged (HiOx-MAP) beef than those in vacuum and polyvinyl chloride (PVC) packaging is not clear. To explore this, the effects of lipid oxidation products with varying carbon chain length on lactate dehydrogenase (LDH) and NADH-dependent metmyoglobin reductase activity were evaluated. Surface color, MRA, and lipid oxidation of beef longissimus lumborum steaks (n = 10) were measured during 6-day display. Further, two enzymes, LDH and NADH-dependent metmyoglobin reductase (n = 5), critical for MRA were incubated with or without (control) lipid oxidation products of varying carbon chain length: malondialdehyde (3-carbon), hexenal (6-carbon), and 4-hydroxynonenal (9-carbon). Steaks in HiOx-MAP had greater (P < 0.05) redness than vacuum and PVC, but had lower (P < 0.05) MRA and greater (P < 0.05) lipid oxidation on day 6. LDH and NADH-dependent metmyoglobin reductase activities were differentially influenced by lipid oxidation products (P < 0.05). The results indicate that the difference in reactivity of various lipid oxidation products on LDH (HNE > MDA = hexenal) and NADH-dependent metmyoglobin reductase (HNE = MDA > hexenal) activity could be responsible for lower MRA in HiOx-MAP.