Proceedings of The Physiological Society

Experimental Models (Exeter, UK) (2018) Proc Physiol Soc 40, C02

Oral Communications

The assembly of a mass spectrometry-based library of the guinea pig proteome as a tool for physiology research

P. Palmowski1, R. Watson1, N. Europe-finner1, A. Treumann2, M. Taggart1

1. Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom. 2. Newcastle University Protein and Proteome Analysis, Newcastle University, Newcastle upon Tyne, United Kingdom.

Recent advances in liquid chromatography-mass spectrometry (LC-MS) approaches have facilitated the incorporation of proteomic studies to many experimental workflows. This has increased our capabilities to assess the changes of hundreds of proteins (abundances or post-translational modifications) occurring with particular insults; in turn, enabling one to interrogate the dynamic proteome-phenotype relationships underlying adaptations to cellular, tissue and organ physiology and pathophysiology challenges. In pursuing experiments to assess broad proteomic profiles from complex cells/tissues, particularly those seeking to take advantage of the high-throughput capabilities and reproducibility of Data-Independent Acquisition LC-MS platforms such as SWATH (Schubert OT et al., 2015), it is desirable to have accessible for reference an extensive background data resource. This is not always readily available if working with species/cells outside of the few very commonly used model systems. For example, the guinea pig is an excellent experimental model for many aspects of human physiology and pathophysiology - including maternal and fetal adapatations to pregnancy (Taggart & Mitchell 2009), cardiac excitation-contraction coupling (Taggart et al., 2014), airway drug responsiveness (Lexmond et al., 2018) and auditory somatosensory processes (Marks et al., 2018) - yet there is limited experimental information regarding the proteome available for this species. In an effort to overcome this obstacle, we sought to generate, via LC-MS/MS measurements, a spectral library of the guinea pig proteome. Homogenates were prepared from 15 tissues (heart, skeletal muscle, brain, uterus, colon, placenta, ovaries, liver, pancreas, lung, kidney, intestines, duodenum, adipose) isolated from sacrificed guinea pigs (fetal- and adult) and each digested to peptides with exposure to trypsin. These tryptic digests were subjected to >200 LC-MS/MS runs to (i) extract peptide-specific information including retention time, m/z value, fragmentation pattern and amino acid sequence; and (ii) thereby, with reference to the guinea pig genome (version: January 2016), identify protein constituents of the proteome. Analysis of >250,000 peptide-spectrum matches resulted in the construction of a library of 74,828 peptides (unique to individual proteins) that corresponded to 7692 proteins. This experimentally validated spectral library increases coverage of the guinea pig proteome >50-fold beyond that publicly available (Uniprot, Dec 2017). It thereby will furnish the research community with a comprehensive proteomic resource to enable (i) exploration of future molecular-phenotypic studies using (re-engaging) the guinea pig as an experimental model and (ii) assessment of cross-species responses to consistent physiological and pathophysiological challenges.

Where applicable, experiments conform with Society ethical requirements