Inconsistent tenderness is one of the most detrimental factors of meat quality. Functional proteomics was used to associate electrophoretic bands from the myofibrillar muscle fraction to meat tenderness in an effort to gain understanding of the mechanisms controlling tenderness. The myofibrillar muscle fraction of the Longissimus dorsi from 22 Angus cross steers was analyzed by SDS-PAGE and linearly regressed to Warner-Bratzler shear values. Six significant electrophoretic bands were characterized by electrophoretic and statistical analysis and sequenced by nano-LC-MS/MS. The protein(s)/peptide(s) identified in these bands encompass a wide array of cellular pathways related to structural, metabolic, chaperone, and developmental functions. This study begins to assemble information that has been reported separately into a more complete picture that will lead to the establishment of a coherent mechanism accounting for meat tenderness.