Preliminary analysis of the proteome of exhaled breath condensate in bottlenose dolphins (Tursiops truncatus)

Authors: BERGFELT, DON - Ross University School Of Veterinary Medicine; Lippolis, John; VANDENPLAS, MICHEL - Ross University School Of Veterinary Medicine; DAVIS, SYDNEY - Ross University School Of Veterinary Medicine; MILLER, BLAKE - Ross University School Of Veterinary Medicine; MADAN, RAHUL - Ross University School Of Veterinary Medicine; KLINE, MAGENTA - Ross University School Of Veterinary Medicine; MARTINEZ, MICHELLE - Dolphin Discovery; SANCHEZ-OKRUCKY, ROBERTO - Dolphin Discovery; DE ALMEIDA, ANDRE - Ross University School Of Veterinary Medicine

Submitted to: Aquatic Mammals
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/4/2018
Publication Date: 5/10/2018
Citation: Bergfelt, D.R., Lippolis, J.D., Vandenplas, M., Davis, S., Miller, B.A., Madan, R., Kline, M., Martinez, M., Sanchez-Okrucky, R., De Almeida, A.M. 2018. Preliminary analysis of the proteome of exhaled breath condensate in bottlenose dolphins (Tursiops truncatus) . Aquatic Mammals. 44/256-266. https://doi.org/10.1578/am.44.3.2018.256.
DOI: https://doi.org/10.1578/am.44.3.2018.256

Interpretive Summary: Blood samples are routinely used to assess health in animals. However, obtaining blood samples in aquatic animals, such as bottlenose dolphins is especially invasive and stressful for the animal and person sampling. It is hypothesized that breath condensate could be a replacement for blood as a means to assess health. The goal of this work was to identify as many proteins in breath condensate as possible as a first step to looking for proteins expression differences that might identify sick dolphins.

Technical Abstract: This primary objective was to conduct an initial analysis of the proteome of exhaled breath condensate or blow in aquarium-based bottlenose dolphins (Tursiops truncatus) and, secondarily, determine the commonality of proteins identified in blow with those in plasma of the same animals as recently documented. Exhaled breath condensate was collected from four young (2 to 6 y), male dolphins using a 50-mL Falcon tube held above the blowhole for 10 sequential exhalations; total volume ranged from 60 to 122 µL. Subsequent to analysis of total protein, 20 µg of protein from each dolphin was separated by SDS-PAGE. Of the 4 samples, 3 produced sufficient resolution of 15 bands that were excised from respective gels and subject to liquid chromatography-tandem mass spectrometry (LC-MS/MS) and used for protein identification. Mass spectra data were used to search the NCBI database restricted to all mammalian proteins. Based on proteins having =2 peptides and present in at least 2 of the 3 dolphins, a total of 220 blow proteins were identified. While a majority (38 to 51%) of proteins could not be categorized, gene ontology indicated protein binding (26%), cytoplasm constituents (16%), and immune response (16%) dominated the molecular function, cellular component location, and biological process domains, respectively. From non-contemporaneous samples, NCBI Accession numbers of 220 blow proteins described herein and 196 plasma proteins previously identified by LC-MS/MS in the same dolphins were matched. Results indicated a commonality of 21 proteins (5%) such that 10 (48%) were related to the immune system (e.g., complement, immunoglobulin-related proteins) and the remainder to other various biological systems. Although preliminary, the novelty of these results provides additional support that exhaled breath condensate can be a relevant, less invasive alternative to blood collection to assess or monitor physiological health and pathological states. More specifically, the commonality of several immune-related proteins between the circulatory and respiratory systems provides a foundation for future investigations to determine the potential of these blow proteins as biomarkers that may be diagnostic or prognostic of respiratory health in bottlenose dolphins and, perhaps, other cetaceans.