Abstract
A data-independent acquisition (DIA) assay library for quantitative analyses of proteome dynamics has been developed for gills of threespine sticklebacks (Gasterosteus aculeatus). A raw spectral library was generated by data-dependent acquisition (DDA) and annotation of tryptic peptides to MSMS spectra and protein database identifiers. The assay library was constructed from the raw spectral library by removal of low-quality, ambiguous, and low-signal peptides. Only unique proteins represented by at least two peptides are included in the assay library, which consists of 1506 proteins, 5074 peptides, 5104 precursors, and 25,322 transitions. This assay library was used with DIA data to identify biochemical differences in gill proteomes of four populations representing different eco- and morpho-types of threespine sticklebacks. The assay library revealed unique and reproducible proteome signatures. Warm-adapted, low-plated, brackish-water fish from Laguna de la Bocana del Rosario (Mexico) show elevated HSP47, extracellular matrix, and innate immunity proteins whereas several immunoglobulins, interferon-induced proteins, ubiquitins, proteolytic enzymes, and nucleic acid remodeling proteins are reduced. Fully-plated, brackish-water fish from Westchester Lagoon (Alaska) display elevated ion regulation, GTPase signaling, and contractile cytoskeleton proteins, altered abundances of many ribosomal, calcium signaling and immunity proteins, and depleted transcriptional regulators and metabolic enzymes. Low-plated freshwater fish from Lake Solano (California) have elevated inflammasomes and proteolytic proteins whereas several iron containing and ion regulatory proteins are reduced. Gills of fully-plated, marine fish from Bodega Harbor (California) have elevated oxidative metabolism enzymes and reduced transglutaminase 2, collagens, and clathrin heavy chains. These distinct proteome signatures represent targets for testing ecological and evolutionary influences on molecular mechanisms of gill function in threespine sticklebacks. Furthermore, the gill assay library represents a model for other tissues and paves the way for accurate and reproducible network analyses of environmental context-dependent proteome dynamics in complex organisms.
Highlights
From the ‡Department of Animal Sciences, University of California Davis, Meyer Hall, One Shields Ave., Davis, CA 95616; §Centro de Investigacion en Alimentacion y Desarrollo, Carretera a la Victoria Km. 0.6, Apartado, Hermosillo, Sonora, Mexico C.P. 83000; ¶Coastal Marine Sciences Institute, University of California, Davis
Gill Assay Library for Ecological Proteomics are suitable for quantitation of more than a thousand proteins simultaneously, which is why they are sometimes referred to as hyper-reaction monitoring (HRM) [10]
For the purpose of this study the DDA data were used for assay library generation and not for systematic comparison of Top3 and spectral counting approaches with assay library quantitation, which exceeds the scope of this article
Summary
From the ‡Department of Animal Sciences, University of California Davis, Meyer Hall, One Shields Ave., Davis, CA 95616; §Centro de Investigacion en Alimentacion y Desarrollo, Carretera a la Victoria Km. 0.6, Apartado, Hermosillo, Sonora, Mexico C.P. 83000; ¶Coastal Marine Sciences Institute, University of California, Davis. Threespine sticklebacks are euryhaline fish that represent a long-standing model in behavioral, evolutionary, and ecological physiology research [11] These fish are ancestrally marine, but the species is represented by many anadromous and land-locked populations that have colonized brackish and freshwater habitats following the retreat of Pleistocene glaciers [12,13,14]. Threespine sticklebacks ideally combine key advantages as a model organism for integrative biology and cross-disciplinary research at the interface of ecophysiology, behavior, evolutionary biology, biochemistry and genetics They are represented by many populations that have naturally adapted to diverse environments, their complete genome has been sequenced, and their proteome is well annotated [14]. The availability of this gill assay library will propel precise, quantitative and network-scale analyses of proteome dynamics in fish experiencing a wide variety of ecological and evolutionary contexts
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