Development and application of an antibody-based protein microarray to assess physiological stress in grizzly bears (Ursus arctos).
A novel antibody-based protein microarray was developed that simultaneously determines expression of 31 stress-associated proteins in skin samples collected from free-ranging grizzly bears (Ursus arctos) in Alberta, Canada. The microarray determines proteins belonging to four broad functional categories associated with stress physiology: hypothalamic-pituitary-adrenal axis proteins, apoptosis/cell cycle proteins, cellular stress/proteotoxicity proteins and oxidative stress/inflammation proteins. Small skin samples (50-100 mg) were collected from captured bears using biopsy punches. Proteins were isolated and labelled with fluorescent dyes, with labelled protein homogenates loaded onto microarrays to hybridize with antibodies. Relative protein expression was determined by comparison with a pooled standard skin sample. The assay was sensitive, requiring 80 µg of protein per sample to be run in triplicate on the microarray. Intra-array and inter-array coefficients of variation for individual proteins were generally <10 and <15%, respectively. With one exception, there were no significant differences in protein expression among skin samples collected from the neck, forelimb, hindlimb and ear in a subsample of n = 4 bears. This suggests that remotely delivered biopsy darts could be used in future sampling. Using generalized linear mixed models, certain proteins within each functional category demonstrated altered expression with respect to differences in year, season, geographical sampling location within Alberta and bear biological parameters, suggesting that these general variables may influence expression of specific proteins in the microarray. Our goal is to apply the protein microarray as a conservation physiology tool that can detect, evaluate and monitor physiological stress in grizzly bears and other species at risk over time in response to environmental change.
Toxicology Graduate Program , University of Saskatchewan , Saskatoon, SK , Canada S7N 5B3.
Ruth I
RI
Carlson
Canadian Wildlife Health Cooperative, Western College of Veterinary Medicine , University of Saskatchewan , Saskatoon, SK , Canada S7N 5B4.
Marc R L
MR
Cattet
Toxicology Centre , University of Saskatchewan , Saskatoon, SK , Canada S7N 5B3.
Bryan L
BL
Sarauer
Department of Renewable Resources , 751 General Services, University of Alberta , Edmonton, AL , Canada T6G 2H1.
Scott E
SE
Nielsen
924 Innes , Integrated Ecological Research , Nelson, BC , Canada V1L 5T2.
John
J
Boulanger
Foothills Research Institute and Alberta Sustainable Resource Development , Hinton, AL , Canada T7V 1V3.
Gordon B
GB
Stenhouse
Department of Veterinary Biomedical Sciences , University of Saskatchewan , Saskatoon, SK , Canada S7N 5B4.
David M
DM
Janz
Grizzly bear; proteomics; skin biopsy; stress