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

Abstract

AbstractQuantitative 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 analyzing 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 non-model organisms. We created a large spectral library composed of 12,553 protein groups from liver and brain tissues. Via identification of differentially expressed proteins (DEPs) under fish exposure to environmental stressors we validated the application and usefulness of these different spectral libraries. Successful identification of significant DEPs from different environmental exposures occurred using the library with a combination of DIA+DDA data as well as both tissue types. Further analysis revealed expected patterns of significantly upregulated 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 coral reef fish and for a non-model organism. It represents a useful guide for the future building of accurate spectral library references in non-model organisms allowing the discovery of ecologically relevant changes in the proteome.