Identification of Proteins from Non-model Organisms Using Mass Spectrometry:  Application to a Hibernating Mammal

Abstract

A major challenge in the life sciences is the extraction of detailed molecular information from plants and animals that are not among the handful of exhaustively studied "model organisms." As a consequence, certain species with novel phenotypes are often ignored due to the lack of searchable databases, tractable genetics, stock centers, and more recently, a sequenced genome. Characterization of phenotype at the molecular level commonly relies on the identification of differentially expressed proteins by combining database searching with tandem mass spectrometry (MS) of peptides derived from protein fragmentation. However, the identification of short peptides from nonmodel organisms can be hampered by the lack of sufficient amino acid sequence homology with proteins in existing databases; therefore, a database search strategy that encompasses both identity and homology can provide stronger evidence than a single search alone. The use of multiple algorithms for database searches may also increase the probability of correct protein identification since it is unlikely that each program would produce false negative or positive hits for the same peptides. In this study, four software packages, Mascot, Pro ID, Sequest, and Pro BLAST, were compared in their ability to identify proteins from the thirteen-lined ground squirrel (Spermophilus tridecemlineatus), a hibernating mammal that lacks a completely sequenced genome. Our results show similarities as well as the degree of variability among different software packages when the identical protein database is searched. In the process of this study, we identified the up-regulation of succinyl CoA-transferase (SCOT) in the heart of hibernators. SCOT is the rate-limiting enzyme in the catabolism of ketone bodies, an important alternative fuel source during hibernation.

Multistate proteomics analysis reveals novel strategies used by a hibernator to precondition the heart and conserve ATP for winter heterothermy

“…2, 11) by a winter abundance increase in ECI1, CRAT, FABP3, and ACADVL (acyl-Coenzyme A dehydrogenase, very long chain) proteins. Additionally, OXCT1 (3-oxoacidCoA transferase 1), which is involved in ketone catabolism, increased in winter, consistent with the study of Russeth et al (57). Thus, our results agree with previous studies that highlight the fundamental importance of lipid metabolism as the main energy-generating source during hibernation.…”

Adaptive mechanisms regulate preferred utilization of ketones in the heart and brain of a hibernating mammal during arousal from torpor

“…Adaptive mechanisms for BHB utilization during hibernation include induction of the BHB transporter MCT1 in the brain (Fig. 3) and a six-fold increase in the level of the rate-limiting enzyme for ketolysis in the heart, succinyl CoA transferase (27). Warmer body temperatures during arousal accelerate BHB uptake from the blood and may explain the reduction in serum BHB levels during IBAs (Fig.…”

Gene expression changes controlling distinct adaptations in the heart and skeletal muscle of a hibernating mammal

“…Substrate utilization in pathological hypertrophy resembles that of the fetal heart, which are characterized by decreased fatty acid oxidation and increased glucose oxidation, while physiological hypertrophy is associated with increased fatty acid and glucose oxidation (59). Evidence from the cardiac transcriptome (19), proteome (83), and metabolic labeling (6) shows that the hibernator heart is increasing fatty acid oxidation and glycolysis without increasing glucose oxidation. The ability of the hibernator to respond to the stress of hibernation with a combination of physiological and pathological hypertrophic responses might be the key to preventing heart failure and maintaining contractile function throughout temperature extremes.…”