Post-mortem (p-m) muscle undergoes a myriad of complex physical and biochemical changes prior to its conversion to meat, which are influential on proteolysis and hence tenderization. A more in-depth understanding of the mechanisms underpinning these dynamics is a key to consistently providing tender beef. Using an LC–MS/MS approach, with state-of-art mass spectrometry Q Exactive HF-X, the proteome and associated pathways contributing to the appearance of the proteolytic breakdown products appearing over 14 days p-m, at two important molecular weights (110 and 30 kDa) on 1D SDS-PAGE gels, have been investigated in beef longissimus thoracis et lumborum muscles exhibiting four rates of pH decline differentiated on the basis of time at pH 6 (fast glycolysing, <3 h; medium, 3–5 h; slow, 5–8 h; and very slow, 8+ h). Both 110 and 30 kDa bands appeared during aging and increased in intensity as a function of p-m time in a pH decline-dependent manner. The 110 kDa band appeared as early as 3 h p-m and displayed an incremental increase in all groups through to 14 days p-m. From 2 days p-m, this increase in abundance during aging was significantly (P < 0.001) influenced by the glycolytic rate: fast > or = medium > slow > very slow. The day 2 p-m appearance of the 30 kDa band was most evident for the fast glycolysing muscle with little or no evidence of appearance in slow and very slow. For days 7 and 14 p-m, the strength of appearance was dependent on glycolysing groups fast > medium > or = slow > very slow. LC–MS/MS analysis yielded a total of 22 unique proteins for the 110 kDa fragment and 13 for the 30 kDa, with 4 common proteins related to both the actin and fibrinogen complex. The Gene Ontology analysis revealed that a myriad of biological pathways are influential with many related to proteins involved primarily in muscle contraction and structure. Other pathways of interest include energy metabolism, apoptotic mitochondrial changes, calcium and ion transport, and so on. Interestingly, most of the proteins composing the fragments were so far identified as biomarkers of beef tenderness and other quality traits.