Seasonal liver protein differences in a hibernator revealed by quantitative proteomics using whole animal isotopic labeling.

Hibernation is an energy-saving strategy used by diverse species of mammals to survive winter. It is characterized by cycles between multi-day periods of torpor with low body temperature (T(b)), and short periods of rapid, spontaneous rewarming. The ability to retain cellular integrity and function throughout torpor and rewarming is a key attribute of hibernation. Livers from winter hibernators are resistant to cellular damage induced by cold storage followed by warm reperfusion. Identifying proteins that differ between the summer-sensitive and winter-protected phenotypic states is one useful approach that may elucidate the molecular mechanisms that underlie this protection. Here we employ a novel quantitative proteomics screening strategy whereby a newly-weaned 13-lined ground squirrel was metabolically labeled by ingesting heavy-isotope substituted ((15)N) Spirulina. The liver protein extract from this animal provided a common reference for quantitative evaluation of protein differences by its addition to extracts from pooled samples of summer active (SA) or winter entrance (Ent) phase hibernating ground squirrels. We identified 61 significantly different proteins between the two groups and compared them to proteins identified previously in the same samples using 2D gels. Of the 20 proteins common to the two datasets, the direction and magnitude of their differences were perfectly concordant for 18, providing confidence that both sets of altered proteins reflect bona fide differences between the two physiological states. Furthermore, the 41 novel proteins recovered in this study included many new enzymes in pathways identified previously: specifically, additional enzymes belonging to the urea cycle, amino acid and carbohydrate degradation, and lipid biosynthetic pathways were decreased, whereas enzymes involved in ketone body synthesis, fatty acid utilization, protein synthesis and gluconeogenesis were increased in the samples from entrance hibernators compared to summer active animals, providing additional specific evidence for the importance of these pathways in the hibernating phenotype.