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High resolution accurate mass spectrometry-based proteomics in ecotoxicology: SWATH-MS to detect differentially expressed plasma proteins in the amphibian toxicological model Xenopus laevis
Citation:
Nelson, K., S. Wilkinson, C. Lavelle, C. Lilavois, S. Raimondo, AND J. Awkerman. High resolution accurate mass spectrometry-based proteomics in ecotoxicology: SWATH-MS to detect differentially expressed plasma proteins in the amphibian toxicological model Xenopus laevis. PRIMO, Charleston, SC, May 19 - 22, 2019.
Impact/Purpose:
To determine the biological effects of chemical exposure in ecotoxicology, there is a need for a complete understanding of molecular changes at the protein level. Discovery of both qualitative and quantitative changes in proteins in plasma or tissues can now be conducted at an unprecedented level using a high resolution accurate mass spectrometer (HRAMS) for data independent analysis (DIA). Our laboratory is building foundational plasma protein ion libraries of the model frog species, Xenopus laevis, to quantitate all protein-level changes across age and sex of individual frogs. Our tissue ion libraries will serve as the basis for determining protein-level changes in X. laevis exposed to chemicals at different life stages. A great advantage to a DIA workflow is the ability to re-analyze data as more genomic and proteomic data becomes publicly available. In the future, our ion libraries can be used by X. laevis researchers, public or private, conducting DIA with HRAMS instrumentation.
Description:
As interest increases in the phenotypic effects of chemical exposure in ecotoxicology, there is a greater need for a complete understanding of corresponding molecular changes. Proteomics data can be used to inform systems toxicology, biomarker discovery, mechanistic toxicology, species extrapolation and adverse outcome pathway development. Until recently, relative quantitative shotgun proteomics experiments required the use of labels, standards, or were susceptible to biased precursor selection. Complex biological samples produced from toxicological exposures, such as plasma or tissue samples, can now be interrogated at an unprecedented depth using a high resolution accurate mass spectrometer with Sequential Window Acquisition of all Theoretical ion fragments (SWATH) to gather a qualitative and quantitative snapshot of all peptides in a sample. SWATH-MS technology uses a multi-part data collection and analysis workflow. First, a complete ion library of every detectable peptide in the sample is created. Next, the library is examined for optimal mass range ionization to create SWATH windows and parameters. Finally, the SWATH data is anchored to the ion library by extracting quantitative information on peptides that meet specific criteria for inclusion. At this point, relative quantitation of all differentially expressed proteins can be examined and compared across samples. A great advantage to this workflow for the field of ecotoxicology is the ability to re-analyze existing data as more genomic and proteomic data becomes publicly available without the need for generating new spectra. For example, our laboratory is building plasma protein ion libraries of the frog, Xenopus laevis, to quantitate protein-level changes between sexes and throughout development. Our current plasma ion library serves as the foundation for evaluating protein-level changes in X. laevis exposed to various chemicals.