Developing deeper understanding of cooking-induced protein modifications in lamb using a proteomic approach

Protein is a key component in meat and contributes significantly to its nutritional and sensory qualities. Typically, consumption of meat is preceded by some form of heat treatment, such as roasting or boiling. While some insight into cooking induced-changes in meat ultrastructure has been reported, at the molecular level much remains to be found out as meat is a highly complex food matrix and is subjected to various kinds of thermal processing conditions. A mass spectrometry-based proteomic approach was employed in this research to explore the protein primary level chemistry of cooking meat. Specifically, mince prepared from 48 h post mortem lamb longissimus thoracis et lumborum was boiled and roasted. In the absence of published in-depth lamb skeletal muscle/meat proteome preceding this study, the proteome of the 48 h post mortem lamb longissimus lumborum pooled from Coopworth lambs was investigated by separating the samples into sarcoplasmic, myofibrillar and insoluble fractions, followed by a shotgun proteomic assessment and bioinformatics analysis. A total of 388 ovine-specific proteins were identified and characterised. More than half of them were found in the sarcoplasmic fraction. The results also verified the existence of at least 300 predicted protein sequences from the sheep genome version 3. With this background proteomic information in hand, the research moved on to examine the changes in protein profile, map and track selected types of amino acid residue-level modifications in response to short (10 min) and long (240 min) periods of hydrothermal treatment (boiling) of the lamb and the corresponding cooking water using a shotgun proteomic approach. Readily distinguishable differences in gel banding patterns and protein identification results of meat and cooking water between the short and the long boiling period were observed. The sodium dodecyl sulphate polyacrylamide gel electrophoresis in conjunction with LC-MS/MS (GeLC) results, specifically for the urea-thiourea extract of cooked meat, revealed that the prolonged boiling caused some proteins to aggregate/polymerise, e.g., glycogen phosphorylase, and some to truncate, e.g., the myosin heavy chains. Several peptides with heat-induced amino acid residue-level modifications such as single oxidation of phenylalanine or carboxyethyllysine, were characterised in the boiling treatment samples. The proteomic approach performed on the boiled lamb meat was then applied to the roasted lamb meat shaped as thin patty in order to minimise the temperature gradient during heating (at 195 ± 5 °C for 5 and 10 min). The 10 min roasting under the given cooking set-up was shown to be much more destructive to protein structure than 240 min boiling as indicated by the drastically reduced protein extractability in the urea-thiourea solution and lack of defined bands in the polyacrylamide gels. Heat-induced amino acid residue-level modifications, e.g., carboxyethyllysine and cyclisation of non-tryptic N-terminal glutamic acid, were characterised in the roasting treatment sample(s). The overall variety of modifications characterised in the roasting treatment samples was less than in the boiling treatment samples. It is likely that certain amino acid residues, particularly the aromatic residues, were modified beyond simple oxidation by the roasting and destroyed completely. This research also showed that actin and myoglobin were the key sources of peptides with heat-induced modification in the cooked meat samples. These peptides could be considered as candidate marker peptides targets for evaluating cooking-induced meat protein modifications across a range of processing conditions. Protein aggregation and alteration of the protein primary strucure can impact on the nutrition and functionality of meat as showed by previous studies. The characterisation of the molecular-level modifications occuring in lamb meat proteins during boiling and roasting by this research provides a good basis for better understanding the effect of these common food processing and preparation methods on the physical and nutritional properties of red meat-based foods