Probing SWATH-MS as a tool for proteome level quantification in a nonmodel fish

Quantitative proteomics via mass spectrometry can provide valuable insight into molecular and phenotypic characteristics of a living system. Recent mass spectrometry developments include data-independent acquisition (SWATH/DIA-MS), an accurate, sensitive and reproducible method for analysing the whole proteome. The main requirement for this method is the creation of a comprehensive spectral library. New technologies have emerged producing larger and more accurate species-specific libraries leading to a progressive collection of proteome references for multiple molecular model species. Here, for the first time, we set out to compare different spectral library constructions using multiple tissues from a coral reef fish to demonstrate its value and feasibility for nonmodel organisms. We created a large spectral library composed of 12,553 protein groups from liver and brain tissues. Via identification of differentially expressed proteins under fish exposure to elevated pCO₂ and temperature, we validated the application and usefulness of these different spectral libraries. Successful identification of significant differentially expressed proteins from different environmental exposures occurred using the library with a combination of data-independent and data-dependent acquisition methods as well as both tissue types. Further analysis revealed expected patterns of significantly up-regulated heat shock proteins in a dual condition of ocean warming and acidification indicating the biological accuracy and relevance of the method. This study provides the first reference spectral library for a nonmodel organism. It represents a useful guide for future building of accurate spectral library references in nonmodel organisms allowing the discovery of ecologically relevant changes in the proteome.