Blood plasma is an attractive source of proteins for biomarker studies in humans and animals. Being a composite of multiple organ specific proteomes, it is paramount in assessing individual’s state of health. Yet, it is one of the most challenging samples for modern mass spectrometry. Recent studies have shown that novel data acquisition strategies, such as SWATH-MS, enable quantitation of hundreds of proteins in non-depleted human plasma. In contrast to human studies however, the SWATH-MS based plasma proteomics in animals is virtually non-existent. Studying animal plasma comes with unique difficulties, such as a lack of protein databases among other challenges. We provide the summary of our 3-year work that resulted in the development of extensive plasma peptide spectral repositories for various domestic animal species, mostly bovids (sheep, cattle), as well as workflows that allow us to reproducibly quantify ~300 proteins in non-depleted plasma using SWATH-MS. We also present a strategy that leverages on the extensive homology between different bovid species to increase the depth of spectral libraries and therefore, increase the number of proteins that can be quantified in plasma. Finally, we present a data analysis pipeline that combines iSwathX, Skyline and in-house scripts to quantify similarly high number of proteins in plasma of non-domestic/exotic ungulate species, for which genome information is not available, and for which building extensive peptide spectral repositories would be unfeasible.
To our knowledge, this is the largest repository of animal plasma proteome as well as the largest number of proteins quantified directly in non-depleted animal plasma. It is also the first example of cross-species utilization of the libraries. Our work has direct implication in wildlife studies where limited access to samples prevents establishing relevant proteomics resources.