High resolution muscle proteome analysis for identification of pH-dependent

Pork technological quality is normally assessed by its color, firmness, and water holding capacity (WHC). A serious problem for final meat quality can be created by a very fast or incomplete pH drop post-mortem. In this way, acceleration of the process of degradation of glycogen from endogenous or exogenous sourses is normally associated with low-quality meat (e.g., PSE and DFD meat). Rapid acidification, when corporal temperature is still high causes the denaturation of proteins. Reduction of solubility and a decrease in water retention means that meat will have a pale, soft, and exudative (PSE) appearance. The opposite phenomenon, when pH does not decrease due to low reserves of glycogen, generates a dark, firm, and dry (DFD) meat with a high water-retention capacity. Based on these quality attributes, pork was traditionally classified into two categories PSE (pale, soft, and exudative), and DFD (dark, firm, and dry). Variation in pork quality has been a major concern since the recognition of the PSE condition. Occurrence of PSE meat is associated with genetic and preslaughter factors that influence post-mortem rate of glycolysis and pH decline. Figure 2.17 shows the pH changes during early post-mortem time [122].

Figure 2.17. Typical pH post-mortem drop of normal, PSE and DFD meats (E. Sayas-Barbera et. al)

For the animals, capture, loading, and transport operations represent a situation of overexcitement and greater muscle activity. All these lead to pre-mortem acceleration in the consumption of ATP and glycogen and the substances free from

degradation of glycogen (CO2, lactic acid), which are forced out of the muscle by the circulatory torrent. When the slaughter of animals is carried out, the muscle has only small quantities of glycogen and there is minimal or no lactic acid production.

Therefore, acidification of meat is low and the drop in pH is incomplete (e.g., not lower than 6.2). These meats have a dry appearance, a closed structure, and are dark red in color [123]. The pH remains high in these meats, which constitutes a risk from the microbiological point of view. These meats constitute a risk, because they are prone to contamination by foodborne pathogens and must be processed carefully, with extreme attention to good hygienic practices [124]. The meat in general has less pH-induced shrinkage (higher water-holding capacity and firmer structure), which leads to a darker color (no denaturation, higher oxygen binding).

DFD-like muscle was observed in the breast and thigh of ducks that had been stressed [125]. On the other hand very fast pH decrease at the prevailing body temperature, more or less above 40 to 42^oC, leads to PSE meat. These characteristics occur as a result of early membrane leakage and protein denaturation, causing shrinkage of fiber leading to the water loss as a drip. PSE is associated with a rapid rate of glycolysis; the rate of glycolysis, leading to a rapid pH fall, which, in combination with high temperature of the muscle, causes protein denaturation [126].

The primary goal of this study was not to generate a detailed 2-DE reference protein map for porcine Longissimus dorsi, but to draw up a tentative map to examine their changes during the post-mortem period according to the pH and drip loss values and to analyse those protein by mass spectrometry. Meat pH value, meat conductivity and meat colour groups were measured using Star-series equipment (Rudolf Matthaeus Company, Germany). Muscle pH, conductivity and meat colour were also measured. Measures were taken at 45 min. post-mortem (pH1ko) and 24 hours post-mortem (pH24) respectively, on the M. Longissimus dorsi between the 13th and 14th ribs (symbol: pH1ko, pH24ko) and in the ham (M. semimembranosus) (symbol: pH24si), respectively. Muscle colour was measured at 24 hours post-mortem by Opto-Star. Drip loss was scored using a bag method by a size-standardised sample from the M. Longissimus dorsi that was collected at 24 hours post-mortem. The sample was weighed, suspended in a plastic bag, held at 4 ^oC for 48 hours and re-weighed at the end of the holding time. Drip loss was calculated as a

percentage of weight loss based on the start weight of a sample. Table 2.4 summarize the muscle sample characteristics taken into study.

Table 2.4. Summary of muscle samples subjected to proteomics analysis Muscle samples ID pH/Drip loss Sex pH/Drip loss value

D049028 pH1KO low Male 5.97

D021042 pH1KO high Female 7.01

D023069 pH24 KO low Male 5.33

D036037 pH24 KO high Male 5.72

D023028 pH24 SI low Female 5.39

D023024 pH24 SI high Male 5.97

D029031 Drip loss low Male 0.8

D022022 Drip loss high Female 5.6

pH1KO - pH value at 45 min. post-mortem in M. Longissimus dorsi pH24 KO - pH value at 24 hrs post-mortem in M. Longissimus dorsi pH24 SI - pH value at 24 hrs post-mortem in M. Semimembranosus

Measurement of pH is used by the pork industry to differentiate product of varying quality. Muscle pH is significantly correlated to attributes such as color and water-holding capacity, which are important characteristics, when consumers make purchasing decisions [127]. Over a wide range of pH (4.86–7.15), Bidner et al. (2004) developed regression equations to describe the relationship of ultimate pH to various quality measurements. Ultimate pH explained 79% of the variation in color, 57% of variation in drip loss, and 77% of the variation in purge loss [128]. Rapid pH decline resulting in ultimate or near ultimate pH while the muscle is still warm causes the denaturation (loss of functionality and water binding ability) of many proteins, including those involved in binding water [129].

The recent development of high throughput proteome analysis equipment allows exploring a large number of muscle proteins simultaneously. However, there are inherent limitations of 2-DE technique, because a few prevailing metabolic enzymes and contractile proteins saturate gel capacity. Approximately 400 µg of total protein solubilized in non-denaturing buffer 2 (s. page 32) extracted from muscle at different post-mortem time and pH values were loaded on IPG strips (3-10) and run in the 1^st dimension. After equilibration, the IPG strips were applied on top of 12 % polyacrylamide gels (Figure 2.18).

Figure 2.18. High resolution 2-D gel electrophoresis of proteins extracted from M. Longissimus dorsi collected 45 min. with high pH (A) and low pH (B), 24 hours with high pH (C) and low pH (D) and 48 hours post-mortem with high drip loss (E) and low drip loss (F).Protein spots showing changes were cut out from the gel, treated with trypsin and, subsequently, analysed by LC-MS/MS.

The gels were stained over night with colloidal Coomassie-blue and scanned using a GS-800 calibrated imaging densitometer (Bio-Rad). Figure 2.18 shows 2-D gels proteome profile of muscle samples collected at 45 min. and 24 hours with different pH and drip loss values. The dominant population of the left side of the 2-D gel was contractile proteins of the actomyosin complex including actin, α- and β-tropomyosin, myosin light chain 1, myosin regulatory light chain 2 and myosin light chain 3. On the other hand, a few metabolic enzymes located on the right side of the 2-D gel, including creatine kinase, β-enolase, glyceraldehydes-3-phosphate, triosephosphate isomerase, myoglobin and hemoglobin were observed. By comparison between the high pH muscle samples (Figure 2.18A and C) and the low pH muscle samples (Figure 2.18B and D), it can be observed myofibrillar protein denaturation at low pH, while metabolic enzymes remained unchanged. No significant difference has been noticed between high pH samples 45 min. and 24 hours, but to some extent myofibrillar proteins were more denatured 24 hours at low pH. A possible explanation of low protein denaturation in low drip samples (Figure 2.18F) is that in this case the pH value is higher and protein denaturation is limited compared with high drip loss (Figure 2.18E) including the proteins involved in water binding. However, differences were observed between M. Longissimus dorsi, where the proteins were found to be denatured only at low pH, while in M.

Semimembranosus, proteins are denatured in both low and high pH (Figure 2.19).

Figure 2.19. High resolution 2-D gel electrophoresis of proteins extracted from M. Semimembranosus collected 24 hours post-mortem at high pH (A) and low pH (B).

The identification of muscle proteins, which showed post-mortem changes according to the pH was performed using LC/MS/MS. Figure 2.20 illustrates mass spectrometric identification of actin located in spot number 1.

Figure 2.20. Identification of actin by LC/MS/MS analyis based on mass fingerprint of 28 peptides covering 59 % of the sequence (shown in red). (A) Total ion chromatogram, (B) MS spectrum of (241-255) tryptic peptide, (C) MS/MS spectrum of m/z 896.3 precursor ion. The very high probability based MOWSE score (328) of protein identification reflects the high number of matched fragment ions.

Initial and ultimate pH can influence the extent of protein denaturation and fresh pork quality attributes such as color and water-holding capacity. Protein denaturation and drip loss increases as muscle ultimate pH decreases. Table 2.5 summarizes the myofibrillar proteins and metabolic enzymes identified by LC/MS/MS.

Table 2.5. Muscle proteins with significant changes at low and high drip loss (high/low pH)

4^a Myosin Regulatory Light

Chain 2 4.8 19 22 84 P97457

a – myofibrillar proteins b – sarcoplasmic proteins

In this study amongst the myofibrillar proteins myosin isoforms were the most affected by the pH. In terms of basic mechanisms, the rate and extent of both pH and temperature fall post-mortem is known the affect the degree of denaturation of the S1 region of myosin heads and to affect the myofilament lattice spacing post-mortem [113]. In genetically stress-susceptible animals, the stress accompanying slaughter, even with carefull handling, is sufficient to trigger a high rate of post-mortem glycolysis. Rigor, i.e., the binding of actin and myosin to form actomyosin, protects myosin against further denaturation. Thus, it is suggested that the rapid rate of post-mortem glycolysis reduces the sensitivity of myosin to denaturation, because the rapid glycolysis results in a rapid rigor. Since glycolysis generates heat, the temperature in a PSE carcass soon after slaughter is higher than before and this will exacerbate the protein denaturation. Another cause of the denaturing conditions responsible for PSE meat occurs if the extent, rather than the rate, of glycolysis is abnormally large [130]. In this case the ultimate pH is low and the muscle proteins are exposed to pHs lower than they normally experience post-mortem. The heads of the native myosin molecule are 19 nm long, but when myosin is heated under conditions resembling that experienced in a PSE carcass, the heads shrink to 17 nm [113].

2.3 Immunological and mass spectrometric characterization of post-mortem