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1. A data-independent acquisition (DIA) assay library for quantitation of environmental effects on the kidney proteome of Oreochromis niloticus

   Root, L; Cnaani, A; Campo, A; MacNiven, L; Kültz, D (January 2021, SICB Virtual Annual Meeting 2021)

   null (Ed.)

   Interactions of organisms with their environment are complex and regulation at different levels of biological organization from genotype to phenotype is often non-linear. While studies of transcriptome regulation are now common for many species, corresponding quantitative studies of environmental effects on proteomes are needed. Here we report the generation of a data-independent acquisition (DIA) assay library that enables simultaneous targeted proteomics of thousands of O. niloticus kidney proteins using a label- and gel-free workflow that is well suited for ecologically relevant field samples. Transcript and protein abundance differences in kidneys of tilapia acclimated to freshwater and brackish water (25 g/kg) were correlated for 2114 unique genes. A high degree of non-linearity in salinity-dependent regulation of transcriptomes and proteomes was revealed, demonstrating the complementary nature of the DIA assay library approach and suggesting that the regulation of O. niloticus renal function by environmental salinity relies heavily on post-transcriptional mechanisms. In addition to significance testing, the application of functional enrichment analyses using STRING and KEGG to DIA assay datasets identified myo-inositol metabolism, antioxidant and xenobiotic functions, and signaling mechanisms as key elements controlled by salinity in tilapia kidneys. In conclusion, this study presents an innovative approach for targeted quantitative proteomics used to identify proteins and biological processes that are regulated non-linearly at mRNA and protein levels during a change of environmental salinity. 

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2. Gill proteome networks explain salinity tolerance of Oreochromis mossambicus

   https://doi.org/10.6069/sgzm-sg20  

   Root, Larken; Kültz, Dietmar (January 2021, Panorama Public)

   Acclimations of Oreochromis mossambicus to elevated salinity were conducted with multiple rates of salinity increase and duration of exposure to determine the rate-independent maximum salinity limit and the incipient lethal salinity. A data-independent acquisition (DIA) assay library was created for quantitative analysis of over 3000 gill proteins simultaneously in treatments representative of important key zones in the salinity level/duration landscape. From these DIA data, protein networks that represent complex molecular phenotypes associated with salinity acclimation were generated. Acclimations of Oreochromis mossambicus to elevated salinity were conducted with multiple rates of salinity increase and duration of exposure to determine the rate-independent maximum salinity limit and the incipient lethal salinity. A data-independent acquisition (DIA) assay library was created for quantitative analysis of over 3000 gill proteins simultaneously in treatments representative of important key zones in the salinity level/duration landscape. From these DIA data, protein networks that represent complex molecular phenotypes associated with salinity acclimation were generated. Protein fold change (FC) and organismal level performance indicators of salinity tolerance were then correlated. Gill protein networks impacted at extreme salinity levels both above and below the incipient lethal limit include increased energy metabolism, especially upregulation of electron transport chain complex proteins, and significant downregulation of a previously uncharacterized protein which bears strong amino acid sequence similarity to fucolectin. Proteins networks strongly impacted by crossing into the zone of resistance include cell adhesion and extracellular matrix regulation. 

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3. Quantitation and comprehension of osmotic effects on proteome dynamics in euryhaline fish

   Kültz, D. (July 2019, SEB 2019 Abstract book)

   Organismal physiology, morphology, and behavior are based on the function of structural proteins and enzymes. Proteins represent the central regulatory plane in the genome to phenome continuum. The protein complement of cells and tissues (the proteome) is highly dynamic and mirrors environmental and developmental influences on organismal phenotypes. Therefore, dynamic proteomes are excellent bioindicators of environmental exposure. Comprehensive blueprints of environmental exposures are reflected in specific proteome states and capturing those states is achieved by quantitative proteomics. We have developed quantitative proteomics workflows to characterize environmental influences on proteome states and proteome dynamics of euryhaline and euryhthermal fish populations in coastal areas. These workflows utilize tissue- and cell-specific assay libraries for data-independent acquisition (DIA) or Sequentially Windowed Acquisition of all THeoretically possible MSMS spectra (SWATH) mass spectrometry. Qunatitative proteome datasets generated with these workflows are highly accurate and they consistently cover precisely defined sets of proteomes. This consistent coverage renders systematic and long-term network and topological data analysis (TDA) approaches feasible. These workflows and approaches are explained and their application to coastal fish biology is discussed using selected datasets as examples. The data presented illustrate that habitat differences such as salinity and temperature changes are readily captured in state changes of tissue-specific proteomes. The overall topology of proteome states is indicative of particular tissues, species, and environmental contexts and is therefore suitable for deducing functional and phenotypic consequences of environmental changes on coastal organisms. 

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4. Deep quantitative proteomics of North American Pacific coast star tunicate

   https://doi.org/10.6069/5fa9-3r35  

   Kültz, Dietmar (January 2023, Panorama Public)

   The first proteomics analysis of a colonial tunicate (Botryllus schlosseri) was carried out to support development of a DIA assay library and quantitative proteomics for this species. Deep proteome coverage (up to 4930 protein groups and 20,984 unique peptides in a single sample) was achieved, which enabled construction of a QC filtered DIA assay library consisting of nearly 4000 proteins that are represented by at least 3 unique peptides, each by at least 4 transitions. This library was used to compare protein abundances of two field populations and one lab-reared B. schlosseri population. Relative protein abundance data indicate that lab-rearing leads to greater proteome changes than those observed between distantly located field populations in Southern California and Northern Washington. STRING analysis revealed functions that are enriched in specific populations and proteins that are least and most variable within a given population. 

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5. Proteomics of Osmoregulatory Responses in Threespine Stickleback Gills

   https://doi.org/10.1093/icb/icaa042  

   Li, Johnathon; Kültz, Dietmar (May 2020, Integrative and Comparative Biology)

   Synopsis The gill proteome of threespine sticklebacks (Gasterosteus aculeatus) differs greatly in populations that inhabit diverse environments characterized by different temperature, salinity, food availability, parasites, and other parameters. To assess the contribution of a specific environmental parameter to such differences it is necessary to isolate its effects from those of other parameters. In this study the effect of environmental salinity on the gill proteome of G. aculeatus was isolated in controlled mesocosm experiments. Salinity-dependent changes in the gill proteome were analyzed by Liquid chromatography/Tandem mass spectrometry data-independent acquisition (DIA) and Skyline. Relative abundances of 1691 proteins representing the molecular phenotype of stickleback gills were quantified using previously developed MSMS spectral and assay libraries in combination with DIA quantitative proteomics. Non-directional stress responses were distinguished from osmoregulatory protein abundance changes by their consistent occurrence during both hypo- and hyper-osmotic salinity stress in six separate mesocosm experiments. If the abundance of a protein was consistently regulated in opposite directions by hyper- versus hypo-osmotic salinity stress, then it was considered an osmoregulatory protein. In contrast, if protein abundance was consistently increased irrespective of whether salinity was increased or decreased, then it was considered a non-directional response protein. KEGG pathway analysis revealed that the salivary secretion, inositol phosphate metabolism, valine, leucine, and isoleucine degradation, citrate cycle, oxidative phosphorylation, and corresponding endocrine and extracellular signaling pathways contain most of the osmoregulatory gill proteins whose abundance is directly proportional to environmental salinity. Most proteins that were inversely correlated with salinity map to KEGG pathways that represent proteostasis, immunity, and related intracellular signaling processes. Non-directional stress response proteins represent fatty and amino acid degradation, purine metabolism, focal adhesion, mRNA surveillance, phagosome, endocytosis, and associated intracellular signaling KEGG pathways. These results demonstrate that G. aculeatus responds to salinity changes by adjusting osmoregulatory mechanisms that are distinct from transient non-directional stress responses to control compatible osmolyte synthesis, transepithelial ion transport, and oxidative energy metabolism. Furthermore, this study establishes salinity as a key factor for causing the regulation of numerous proteins and KEGG pathways with established functions in proteostasis, immunity, and tissue remodeling. We conclude that the corresponding osmoregulatory gill proteins and KEGG pathways represent molecular phenotypes that promote transepithelial ion transport, cellular osmoregulation, and gill epithelial remodeling to adjust gill function to environmental salinity. 

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