Development of a Gill Assay Library for Ecological Proteomics of Threespine Sticklebacks (Gasterosteus aculeatus)*

A tissue-specific assay library for quantitative proteomics by DIA LC-MS/MS has been developed for threespine stickleback gills. The utility of this assay library for discriminating proteomes from four stickleback populations representing different ecotypes has been demonstrated. The results reported reveal that this assay library provides comprehensive insight into population-specific stickleback gill proteomes. This insight informs targeted studies on mechanisms of adaptation to extreme habitats. Graphical Abstract Highlights Construction of threespine stickleback gill assay library using DDA proteomics Population-specific gill proteome signatures of four ecotypes identified by DIA HSP47 and extracellular matrix proteins highly elevated in warm-adapted sticklebacks Inflammasome and proteolytic proteins highly elevated in freshwater sticklebacks 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.