This proteomics screen was designed to bypass the use of 2D gels to quantify liver protein differences between SA and Ent hibernators. Quantification was achieved by calculating the relative ratio of natural abundance peptides to their heavy-isotope counterparts in a common reference standard (). Liver extracts were used for several reasons: liver plays a central role in metabolic regulation; the same livers were analyzed previously using a 2D gel approach incorporating internal standards and CyDye labeling (Epperson et al., 2010
) allowing a direct comparison of the protein similarities and differences revealed by the two methods; results from two previous proteomic screens of liver extracts from related hibernators, golden-mantled (Epperson et al., 2004
) and arctic (Shao et al., 2010
) ground squirrels, are also available and provide additional datasets for comparison. By avoiding 2D gels in this study we hoped to increase the numbers of membrane proteins evaluated between these two dramatically different physiological states.
Fig. 2 Schematic of experimental strategy. Leftmost squirrel was metabolically labeled by feeding 15N-Spirulina. Liver protein from this animal was mixed with an equal amount of liver protein from a pool of either 6 SA or 6 Ent ground squirrels, and the proteins (more ...)
The internal reference standard labeled with 15
N comprised total liver protein extracted from a young 13-lined ground squirrel fed a special diet for 4 weeks immediately after weaning in which all nitrogen came from 15
. Ground squirrels fed the 15
N diet grew to the same body weight as their littermates fed a normal diet; no differences were noted in behavior, appearance, or growth rates as assessed by body weight (). In the 4-week labeling period, the ground squirrels approximately doubled their body weight. Liver histology was normal () in the presence of 15
N, as reported previously for rats (Wu et al., 2004
). Based on GC/MS analysis of hydrolyzed liver proteins in each of the two animals fed the heavy diet, the average 15
N enrichment in amino acids was 92.65 and 92.98%, respectively.
Fig. 3 Normal weight gain of ground squirrels fed 15N. Three animals were fed protein-free rat chow supplemented with Spirulina from weaning (day 0). Animal 2055 (open squares) received Spirulina containing natural abundance nitrogen isotopes, whereas the Spirulina (more ...)
Fig. 4 Normal liver morphology in Spirulina-fed 13-lined ground squirrels. Representative micrographs of liver sections from A) 15N-enriched, or B) conventional (14N) Spirulina control as the only source of dietary nitrogen, stained with hematoxylin and eosin. (more ...)
N reference was mixed into a pooled sample containing equal amounts of protein extract prepared from six individual SA livers, and another sample containing a mixture from six Ent livers. These mixtures were fractionated by SDS-PAGE to increase the probability that multiple peptides corresponding to a single protein would be recovered. After electrophoresis, each gel lane, containing either the pooled SA or Ent sample spiked with the 15
N reference standard, was excised and cut into 88 2mm sections. Each gel slice therefore contained a complex, size-restricted mixture of liver proteins. These gel sections were individually processed to release peptides, and peptide mixtures were analyzed by multi-dimensional protein identification technology (MuDPIT, , Washburn et al., 2002
; Wu et al., 2003
). We recovered 652 and 566 non-redundant orthologous protein groups from the SA and Ent gel lanes, respectively, after filtering for contaminants and redundancy as described in section 2.6. 389 of these groups were matched between seasons on the basis of shared peptides and species-truncated protein name. Of these, RelEx analysis revealed that 61 distinct liver proteins differed significantly (p<0.05) between the two states: 27 were increased in Ent hibernators () and 34 were decreased () relative to SA.
These same Ent and SA liver protein extracts were used previously to screen for protein differences using a quantitative 2D gel method (Epperson et al., 2010
), providing a unique opportunity to compare the proteins recovered using the two techniques. Although the majority of protein differences between Ent and SA livers were revealed by just one of the two methods (), the magnitude and direction of change of the 20 proteins recovered by both methods were highly concordant, lending validity to both methods. Specifically, 18 of the 20 were perfectly concordant for both datasets, with just two differing in the direction of change ().
Fig. 5 Comparison of liver protein differences between SA and Ent hibernators as revealed by DiGE and stable isotope quantification. A. Venn diagram depicting unique proteins that differ between Ent and SA that were revealed by each method or by both methods. (more ...)
This proteomics screening approach was specifically undertaken to try to enhance recovery and assessment of membrane proteins by avoiding the use of isoelectric focusing, which is not well-suited for analysis of membrane proteins (Santoni et al., 2000
). Twelve of the proteins recovered in this study were annotated to the GO cellular component category of organelle membrane and nine of these were contained in a subset annotated as mitochondrial inner membrane using the DAVID webserver (Dennis et al., 2003
; Huang et al., 2009
). However, database annotations regarding membrane association are often ambiguous, failing to distinguish membrane-associated proteins from true integral membrane proteins. Approximately 20–30% of cellular proteins are thought to be integral membrane proteins (Krogh et al., 2001
; Stevens and Arkin, 2000
), i.e., proteins that contain membrane spanning helices, which can be reliably predicted from primary sequence data (Krogh et al., 2001
) using a web server tool (http://www.cbs.dtu.dk/services/TMHMM/
). The proteins listed in and were examined for membrane spanning helices using TMHMM. Of the 61 uniquely identified proteins in this study, six are predicted to contain membrane spanning helices: protein disulfide isomerase-related protein (PDIA6), cyclophilin B (PPIB), oxygen regulated protein precursor (HYOU1), acyl-CoA synthestase long-chain 5 (ACSL5), aldehyde dehydrogenase 6A1 (ALDH6A1), and nicotinamide nucleotide transhydrogenase (NNT). The latter protein contains 12 predicted transmembrane domains, whereas the other proteins each contain only one. Thus, NNT is likely the only transmembrane protein recovered in this analysis that theoretically could not have been found using 2D gels, which generally fail to resolve proteins with two or more transmembrane domains (Kline and Wu, 2009
). None of the six proteins with predicted TM domains are among the list of proteins recovered by both methods (), however three of the proteins recovered by DiGE: SLC27A2, CYB5A and ACMSD, are predicted to have TM domains using TMHMM. In the present study, and combined contain 61 unique proteins; therefore, if no biases prevent membrane proteins from changing as a function of hibernation status, and they were efficiently recovered by this method that does not rely on in-gel isoelectric focusing, 12–18 transmembrane-containing proteins would be expected.
To assess whether the limited number of membrane proteins recovered in this screen for protein differences between Ent and SA livers was simply a reflection of an inefficiency of the method in recovering membrane proteins, or if it instead represents a bias against membrane protein changes depending upon hibernation status, the complete lists of proteins identified in the present study were also analyzed by TMHMM. TM domains were predicted in 72 (of 652, 11.0%) and 43 (of 566, 7.6%) of the proteins in the SA and Ent lists, respectively. Both lists again had substantially fewer than the expected 20–30% of TM domain-containing proteins, indicating that a bias against recovery of proteins with TM domains simply propagated to the list of proteins that differed between the two hibernation states. Thus, from the available data it is most reasonable to conclude that proteins with TM domains are as likely to differ between SA and Ent animals as those without TM domains and that membrane proteins are underrepresented in the proteins that can be evaluated by this method.
A gene enrichment analysis was performed on the lists of proteins that were increased or decreased in Ent livers using DAVID. With the list of all liver proteins that were recovered in this experiment as background, the proteins that increased during Ent were enriched in processes of fatty acid metabolism (mainly catabolism), regulation of apoptosis (anti-apoptosis) and cellular redox homeostasis. Over-represented cellular components were endoplasmic reticulum and ribosome (). In contrast, the proteins increased in SA were involved with catabolism of nitrogen, ATP and nucleotides, and energy derived from the oxidation of organic compounds. They were also enriched in mitochondrial and lysosomal proteins (). When the human database was used as the background for the DAVID analysis instead, similar results were obtained, except that glucose catabolism became significantly enriched in SA (not shown). These findings strongly support previous results demonstrating a substantial change in metabolic and biosynthetic priorities between livers from Ent and SA 13-lined ground squirrels (Epperson et al., 2010
Enriched DAVID annotation categories in the list of proteins that increased in Ent
Enriched DAVID annotation categories in the list of proteins that decreased in Ent