Rapid Peptidome Enrichment Coupled With Label-Free Quantification to Characterize Proteome Degradation and Proteolytic Cleavage in Postmortem Beef Longissimus Lumborum Muscle

There is limited understanding of proteolytic cleavage during protein degradation in the scope of the whole proteome, primarily because the typical bottom-up proteomics workflow ignores atypical proteolysis patterns of endogenous proteins targeted for degradation. For the first time, the current study demonstrated an easy-to-prepare, low-mass spectrometry running cost and accurate nontargeted peptidomic workflow as well as multidimensional quantitative analyses to characterize proteome degradation and proteolytic cleavage in bovine longissimus lumborum (n = 8) from 2 h to 48 h and 48 h to 336 h postmortem. Four-dimensional quantitative analyses were conducted on 3860 quantified endogenous peptides, 244 precursor proteins, 4702 cleavage sites, and 363 cleavage residues in P1-P1’ positions. A total of 81 precursor proteins (P < .05) were identified with significantly increased degradation in each or both postmortem periods, including known degradation of many proteins contributing to tenderization, such as desmin, troponin T, and nebulin, and newly identified degradations of many cellular structure proteins, such as myotilin, myozenin, telethonin, myomesin, vimentin, synemin, and β-actin. Increased degradation of glycolytic enzymes (e.g., phosphofructokinase-1, glyceraldehyde-3-phosphate dehydrogenase, and L-lactate dehydrogenase A chain), mitochondrial proteins (complex I and IV), and meat color-related proteins (myoglobin and flavin reductase) were also identified for the first time. Increased overall peptide abundance indicated that postmortem proteolysis is mainly located at the myofibril and cytoskeleton, with a growing contribution from glycolytic enzymes during postmortem aging. Cleavage-site analysis documented known and unknown cleavage sites of protein degradation at postmortem, including the canonical and atypical cleavages of calpain-1 and calpain-3. Cleavage residue analysis indicated glutamate-alanine and histidine-glutamate were 2 peptide bonds with the most abundant cleavage during both postmortem periods, while the cleavage after lysine was placed with residues with different polarity and hydrophobicity. The demasking and secondary masking of proteolytic sites/residues during proteome degradation in the 2 postmortem periods were also demonstrated. The workflow and analytical strategy presented by the current study can be applied extensively to identify the key cleavage events that affect meat quality development.