The challenge of proteomics, the next frontier of biological and biomedical research, is essentially based on archetypal “Analytical Chemistry” strategies: separation by electrophoresis techniques, identification and quantitative analysis through mass spectrometry. Studying the proteome expressed in mussel tissues is important to understand many different biological, physiological and ecological aspects that may be of advantage in shellfish rearing, encompassing growth cycle, food safety and traceability as well as understanding the biological effects of environmental pollution and climate change. The present work attempted to accomplish a systematic characterization of the foot muscle proteome of M. galloprovincialis, to gather data about its variability in physiological conditions occurring in different environments of mussel rearing and to identify a putative set of protein biomarkers that may be significantly correlated to environmental factors. A total of 360 mussels were sampled from three different lagoons of Sardinia, each one characterized by its different environmental and ecological system. Classical and multivariate statistics was performed to study the protein expression data and to correlate the expression levels of the proteins of interest (79 identified protein) to the content of trace metals in mussels and environmental parameters recorded in situ.