LC–MS/MS Quantification of Plasma Proteins in Elephant Seals to Advance Wildlife Conservation

Animals in the wild face a range of stressors, many of which are recurring or chronic. Stress hormone levels in animals are highly context-dependent and do not consistently predict chronic stress in wildlife. However, profiling of downstream consequences of repeated stress responses (for example, changes in metabolism or gene expression) may be more informative when predicting an animal’s individual-level health consequences and population-level impacts, both key objectives for wildlife conservation.

A joint study by the University of the Pacific (Stockton, CA), the National Marine Mammal Foundation (San Diego, CA) and Sonoma State University (Rohnert Park, CA) examined the plasma proteome response of a quartet of northern elephant seals (NES; Mirounga angustirostris) to a repeated stress experiment, since multiple organs secrete proteins into the circulation, providing a readout of their activity and integration. Plasma proteins were isolated, identified, and quantified using liquid chromatography and tandem mass spectrometry (LC-MS/MS), and their abundance was compared between sampling times. The research team identified more than 200 proteins in plasma, 42 of which were altered in abundance, revealing complex protein dynamics in response to repeated stress challenges. A paper detailing these findings was recently published in Conservation Physiology (1).

Members of the team were co-authors on a paper describing an experiment which simulated repeated stress in juvenile NES using adrenocorticotropic hormone (ACTH) administration (2).

That life history stage was chosen by the researchers because the NES were not yet molting, reproducing, or fasting extensively during this time, and did not yet demonstrate associated variation in baseline corticosteroid and thyroid hormone levels (3,4). Cortisol responses to each of four ACTH challenges administered ~24 h apart were highly variable between the seals dosed and did not significantly differ between the first and fourth days. Aldosterone and total T3 levels, as well as the transcriptome and proteome of blubber tissue, were significantly altered by repeated ACTH challenges, and yielded additional markers (for example, hormones other than GCs, genes associated with lipolysis, and adipogenesis) for distinguishing responses to single versus multiple stressors (5-7).

The blubber proteome response to ACTH, however, was limited to few proteins primarily associated with connective tissue function, which provided incomplete insights into the functional consequences of repeated stress responses (7). As previous work some of the authors of the most recent paper revealed that the plasma proteome was more responsive to prolonged physiological challenges than blubber or muscle (8), suggesting that this matrix is a rich source of biomarkers, a hypothesis was formed that the plasma proteome would reflect integration of tissue responses to ACTH administration, thus providing valuable information about the indicators and impacts of recent and repeated stress in marine mammals.

Delayed, but sustained, changes in the plasma proteome in response to repeated ACTH administration in the NES were identified by the researchers. High individual variability in animal responses to ACTH precluded identification of immediate, short-term changes in the plasma proteome, which may become apparent in a larger study. The findings suggest that, unlike the transcriptional and proteomic responses of individual tissues, such as blubber, to ACTH (which were detectable within several hours of administration), the plasma proteome may be more suitable for examining longer-term integration of multi-organ responses to recent, rather than immediate, challenges (9). The high temporal resolution provided by repeated sampling over four days in this study offered a unique opportunity to examine proteome dynamics and multi-phase responses to the experiment (for example, immediate vs. second- and third-order responses) (1).

Two male bull elephant seals on the beach in Central California. © shielaf2002 - stock.adobe.com

References

1. Avalos, J. G.; Champagne, C. D.;Crocker, D.E.; Khudyakov, J. I. The Plasma Proteome Reveals Markers of Recent and Repeated Stress in Free-Ranging Seals. Conserv. Physiol. 2024, 12 (1), coae075. DOI: 10.1093/conphys/coae075

2. McCormley, M. C.: Champagne, C. D.; Deyarmin, J. S.; Stephan, A. P.; Crocker, D. E.;Houser, D. S.; Khudyakov, J. I. (2018). Repeated adrenocorticotropic hormone administration alters adrenal and thyroid hormones in free-ranging elephant seals. Conserv. Physiol. 2018, 6 (1), coy040. DOI: 10.1093/conphys/coy040

3. Kelso, E. J.; Champagne, C. D.; Tift, M. S.; Houser, D. S.; Crocker, D. E. Sex Differences in Fuel Use and Metabolism During Development in Fasting Juvenile Northern Elephant Seals. J. Exp Biol. 2012,215 (Pt 15), 2637–2645. DOI: 10.1242/jeb.068833

4. Jelincic, J. A.; Tift, M. S.; Houser, D. S.; Crocker, D. E. Variation in Adrenal and Thyroid Hormones with Life-History Stage in Juvenile Northern Elephant Seals (Mirounga angustirostris). Gen. Comp. Endocrinol. 2017, 252, 111–118. DOI: 10.1016/j.ygcen.2017.08.001

5. McCormley, M. C.; Champagne, C. D.; Deyarmin, J. S.; Stephan, A. P.; Crocker, D. E.; Houser, D. S.; Khudyakov, J. I. Repeated Adrenocorticotropic Hormone Administration Alters Adrenal and Thyroid Hormones in Free-Ranging Elephant Seals, Conserv, Physiol. 2018, 6 (1), coy040. DOI: 10.1093/conphys/coy040

6. Deyarmin, J. S.; McCormley, M. C.; Champagne, C. D.; Stephan, A. P.; Busqueta, L, P.; Crocker, D. E.; Houser, D. S.; Khudyakov, J. I. Blubber Transcriptome Responses to Repeated ACTH Administration in a Marine Mammal. Sci. Rep. 2019,9 (1), 2718. DOI: 10.1038/s41598-019-39089-2

7. Deyarmin, J.; Hekman, R.; Champagne, C.; McCormley, M.; Stephan, A.; Crocker, D. et al. Blubber Proteome Response to Repeated ACTH Administration in a Wild Marine Mammal. Comp. Biochem. Physiol., Part D: Genomics Proteomics2020, 33, 100644. DOI: 10.1016/j.cbd.2019.100644

8. Khudyakov, J. I.; Holser, R. R.; Vierra, C. A.; Ly, S. T.; Niel, T. K.; Hasan, B. M.; Crocker, D. E.; Costa, D. P. Changes in Apolipoprotein Abundance Dominate Proteome Responses to Prolonged Fasting in Elephant Seals. J. Exp. Biol. 2022, 225 (4), jeb243572. DOI: 10.1242/jeb.243572

9. Gormally, B. M.; Romero, L. M. What Are You Actually Measuring? A Review of Techniques that Integrate the Stress Response on Distinct Time‐Scales. Funct. Ecol. 2020, 34(10), 2030–2044. DOI: 10.1111/1365-2435.13648