24 Evaluation of an antibody-free approach to identifying faecal peptides for pregnancy detection in polar bears (Ursus maritimus)

The ability to diagnose pregnancy would have beneficial applications for monitoring bear populations both in situ and ex situ, but currently there is no method of noninvasive pregnancy detection in Ursidae. Recent reports in several carnivore species have described the identification of faecal proteins that may serve as pregnancy biomarkers; however, repeatability has been limited. It is possible that antibody-based methods of protein detection, such as ELISAs, are unsuitable for quantifying faecal proteins because of protein degradation inherent to the sample matrix that likely affects antibody-binding sites and thus precludes accurate quantification. The objective of the current analysis was to evaluate an unbiased, antibody-free, label-free method for the identification and quantification of protein components of faecal samples to determine whether differences associated with pregnancy are detectable. All samples were collected noninvasively from zoo-housed polar bears from 2011 to 2016 and stored frozen. Protein was extracted from frozen-thawed samples (n = 48) obtained from parturient (n = 6) and nonparturient (n = 6) bears at four timepoints: pre-breeding season (26 Jan ± 3.5), embryonic diapause (1 Aug ± 0.5), early placental pregnancy (43–48 days prepartum; 21 Oct ± 3.9), and mid-placental pregnancy (30–32 days prepartum; 7 Nov ± 3.7). For the non-parturient group, sample dates were matched to the parturient group. Additionally, samples were age-matched to control for potential effects of storage time on protein degradation. Briefly, equal amounts of protein (2.5 ug) from each sample were gel-separated, excised, trypsin digested, and analysed on the Thermo Orbitrap Eclipse nanoLC-MS/MS system (ThermoFisher Scientific) using a 90-min label-free quantitation (LFQ) workflow. The dataset was searched against a combined Ursus species protein database with Proteome discoverer ver 2.4 and the Sequest HT search algorithm (ThermoFisher Scientific) with LFQ parameters. In total, 312 proteins were identified and quantified. Of those, 191 (61.2%) were detected across all samples and 123 showed >2-fold change in the mean protein abundance ratio between parturient and nonparturient groups for at least one timepoint; however, coefficients of variation were high for both abundance ratio variability (384.8 ± 61.0% s.e.m.) and within-group variability (86.8 ± 1.5%) by timepoint, indicating low reproducibility and inconsistency of individual protein abundances in grouped samples. Results of this study suggest that the inconsistencies in specific protein concentrations revealed previously by antibody-based assays may not be due to that methodology’s limitations, but rather reflect the true variation that exists among samples. It is possible that low sample numbers, differences in and duration of storage conditions, diverse diets, inconsistent gut passage time, and/or sample degradation before storage all contributed to the variability in protein abundances observed in the current study. These findings highlight the challenges of faecal protein analyses in wildlife, especially when samples are collected opportunistically from heterogenous populations.

This project was made possible in part by the Institute of Museum and Library Services grant #MA-30-18-0461-18.