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

   https://doi.org/10.1111/1755-0998.13445  

   Root, Larken ; Campo, Aurora ; MacNiven, Leah ; Con, Pazit ; Cnaani, Avner ; Kültz, Dietmar ( June 2021 , Molecular Ecology Resources)

   Interactions of organisms with their environment are complex and environmental regulation at different levels of biological organization is often nonlinear. Therefore, the genotype to phenotype continuum requires study at multiple levels of organization. While studies of transcriptome regulation are now common for many species, 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 ofOreochromis niloticuskidney proteins using a label‐ and gel‐free workflow that is well suited for ecologically relevant field samples. We demonstrate the usefulness of this DIA assay library by discerning environmental effects on the kidney proteome ofO. niloticus. Moreover, we demonstrate that the DIA assay library approach generates data that are complimentary rather than redundant to transcriptomic data. 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 suggesting that the regulation ofO.niloticusrenal function by environmental salinity relies heavily on post‐transcriptional mechanisms. The application of functional enrichment analyses using STRING and KEGG to DIA assay data sets is demonstrated by identifyingmyo‐inositol metabolism, antioxidant and xenobiotic functions, and signalling mechanisms as key elements controlled by salinity in tilapia kidneys. The DIA assay library resource presented here can be adopted for other tissues and other organisms to study proteome dynamics during changing ecological contexts.

    

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3. 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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4. 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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5. A comprehensive spectral assay library to quantify the Halobacterium salinarum NRC-1 proteome by DIA/SWATH-MS

   https://doi.org/10.1038/s41597-023-02590-5  

   Kusebauch, Ulrike ; Lorenzetti, Alan P. R. ; Campbell, David S. ; Pan, Min ; Shteynberg, David ; Kapil, Charu ; Midha, Mukul K. ; López García de Lomana, Adrián ; Baliga, Nitin S. ; Moritz, Robert L. ( October 2023 , Scientific Data)

   Data-Independent Acquisition (DIA) is a mass spectrometry-based method to reliably identify and reproducibly quantify large fractions of a target proteome. The peptide-centric data analysis strategy employed in DIA requiresa priorigenerated spectral assay libraries. Such assay libraries allow to extract quantitative data in a targeted approach and have been generated for human, mouse, zebrafish,E. coliand few other organisms. However, a spectral assay library for the extreme halophilic archaeonHalobacterium salinarumNRC-1, a model organism that contributed to several notable discoveries, is not publicly available yet. Here, we report a comprehensive spectral assay library to measure 2,563 of 2,646 annotatedH. salinarumNRC-1 proteins. We demonstrate the utility of this library by measuring global protein abundances over time under standard growth conditions. TheH. salinarumNRC-1 library includes 21,074 distinct peptides representing 97% of the predicted proteome and provides a new, valuable resource to confidently measure and quantify any protein of this archaeon. Data and spectral assay libraries are available via ProteomeXchange (PXD042770, PXD042774) and SWATHAtlas (SAL00312-SAL00319).

    

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