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

ABSTRACT: 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.

SUBMITTER: Monroe AA

PROVIDER: S-EPMC7689905 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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

Monroe Alison A AA Zhang Huoming H Schunter Celia C Ravasi Timothy T

Molecular ecology resources 20200804 6

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 leadi ...[more]

PMID: 32687632

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Project description:Targeted mass spectrometry by selected reaction monitoring (S/MRM) has proven to be a suitable technique for the consistent and reproducible quantification of proteins across multiple biological samples and a wide dynamic range. This performance profile is an important prerequisite for systems biology and biomedical research. However, the method is limited to the measurements of a few hundred peptides per LC-MS analysis. Recently, we introduced SWATH-MS, a combination of data independent acquisition and targeted data analysis that vastly extends the number of peptides/proteins quantified per sample, while maintaining the favorable performance profile of S/MRM. Here we applied the SWATH-MS technique to quantify changes over time in a large fraction of the proteome expressed in Saccharomyces cerevisiae in response to osmotic stress. We sampled cell cultures in biological triplicates at six time points following the application of osmotic stress and acquired single injection data independent acquisition data sets on a high-resolution 5600 tripleTOF instrument operated in SWATH mode. Proteins were quantified by the targeted extraction and integration of transition signal groups from the SWATH-MS datasets for peptides that are proteotypic for specific yeast proteins. We consistently identified and quantified more than 15,000 peptides and 2500 proteins across the 18 samples. We demonstrate high reproducibility between technical and biological replicates across all time points and protein abundances. In addition, we show that the abundance of hundreds of proteins was significantly regulated upon osmotic shock, and pathway enrichment analysis revealed that the proteins reacting to osmotic shock are mainly involved in the carbohydrate and amino acid metabolism. Overall, this study demonstrates the ability of SWATH-MS to efficiently generate reproducible, consistent, and quantitatively accurate measurements of a large fraction of a proteome across multiple samples.

Dataset Information

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

Publications

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

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