Dataset Information

Nonlinear effects of environmental salinity on the gill transcriptome versus proteome of Oreochromis niloticus

ABSTRACT: Here we report the generation of a data-independent acquisition (DIA) assay library that enables simultaneous targeted proteomics of 1900 O. niloticus gill proteins using a label- and gel-free workflow that is well suited for ecologically relevant field samples. By determining alignment and mismatch between protein and mRNA regulation, the DIA assay library approach generates data that are complimentary rather than redundant to transcriptomics data. Transcript and protein abundance differences in gills of tilapia acclimated to freshwater and brackish water (25 g/kg) revealed non-linearity in salinity-dependent transcriptome versus proteome regulation. Non-linearity was more evident for specific functional groups of genes while other molecular functions/ cellular processes where more highly correlated regarding mRNA and protein regulation. Our study identifies specific salinity-dependent O. niloticus gill functions and processes that rely heavily on mRNA abundance regulation and others that rely more heavily on regulatory mechanisms beyond the transcriptome level. The DIA assay library approach presented here is shown to be a powerful means of complementing transcriptome data with corresponding quantitative proteome data to better discern mechanisms of regulation along the genome to phenome continuum.

ORGANISM(S): Oreochromis Niloticus

SUBMITTER: Dietmar Kültz

PROVIDER: PXD024648 | panorama | Sat Oct 23 00:00:00 BST 2021

REPOSITORIES: PanoramaPublic

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Publications

Nonlinear effects of environmental salinity on the gill transcriptome versus proteome of Oreochromis niloticus modulate epithelial cell turnover.

Root Larken L Campo Aurora A MacNiven Leah L Con Pazit P Cnaani Avner A Kültz Dietmar D

Genomics 20210720 5

A data-independent acquisition (DIA) assay library for targeted quantitation of thousands of Oreochromis niloticus gill proteins using a label- and gel-free workflow was generated and used to compare protein and mRNA abundances. This approach generated complimentary rather than redundant data for 1899 unique genes in gills of tilapia acclimated to freshwater and brackish water. Functional enrichment analyses identified mitochondrial energy metabolism, serine protease and immunity-related functio ...[more]

PMID: 34298068

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Project description:The effect of environmental salinity on the gill proteome was isolated in controlled mesocosm experiments. Salinity-dependent changes in the gill proteome were analyzed by LCMSMS data-independent acquisition (DIA) and Skyline. For this purpose, 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 data-independent acquisition (DIA) quantitative proteomics (Li et al. 2018). Osmoregulatory responses were distinguished from general stress responses based on the direction of protein abundance changes. If the abundance of a protein was consistently regulated in opposite directions by hyper- versus hypo-osmotic salinity stress in all six mesocosm experiments then it was considered an osmoregulatory protein. If protein abundance was always increased in all six mesocosm experiments independent of whether salinity was increased or decreased then it was considered a stress response protein. KEGG pathway analysis revealed that the inositol phosphate metabolism, salivary secretion, several endocrine and extracellular signaling, valine, leucine and isoleucine degradation, citrate cycle, and oxidative phosphorylation pathways contain most osmoregulatory gill proteins whose abundance is directly proportional to environmental salinity. Most proteins that were inversely correlated with salinity map to pathways that represent proteostasis, immunity and intracellular signaling processes. General stress response proteins represent fatty and amino acid degradation, purine metabolism, focal adhesion, mRNA surveillance, phagosome, endocytosis, and several intracellular signaling pathways. Some proteins in these general stress response pathways are consistently down-regulated instead of being up-regulated during any type of salinity stress indicating that these KEGG pathways are not uniformly induced or repressed but that their function is being altered more intricately.

Project description:Data independent acquisition-mass spectrometry (DIA-MS) coupled with liquid chromatography is a promising approach for rapid, automatic sampling of MS/MS data in untargeted metabolomics. However, wide isolation windows in DIA-MS generate MS/MS spectra containing a mixed population of fragment ions together with their precursor ions. This precursor-fragment ion map in a comprehensive MS/MS spectral library is crucial for relative quantification of fragment ions uniquely representative of each precursor ion. However, existing reference libraries are not sufficient for this purpose since the fragmentation patterns of small molecules can vary in different instrument setups. Here we developed a bioinformatics workflow called MetaboDIA to build customized MS/MS spectral libraries using a user's own data dependent acquisition (DDA) data and to perform MS/MS-based quantification with DIA data, thus complementing conventional MS1-based quantification. MetaboDIA also allows users to build a spectral library directly from DIA data in studies of a large sample size. Using a marine algae data set, we show that quantification of fragment ions extracted with a customized MS/MS library can provide as reliable quantitative data as the direct quantification of precursor ions based on MS1 data. To test its applicability in complex samples, we applied MetaboDIA to a clinical serum metabolomics data set, where we built a DDA-based spectral library containing consensus spectra for 1829 compounds. We performed fragment ion quantification using DIA data using this library, yielding sensitive differential expression analysis. </br></br> Serum metabolome of 40 age-related macular degeneration patients and 20 control samples was analyzed using untargeted mass spectrometry. We used data dependent acquisition data to build a MS/MS spectral assay library for more than 1,000 compounds and performed targeted extraction of MS2 ion chromatograms from data independent acquisition analysis.

Dataset Information

Nonlinear effects of environmental salinity on the gill transcriptome versus proteome of Oreochromis niloticus

Publications

Nonlinear effects of environmental salinity on the gill transcriptome versus proteome of Oreochromis niloticus modulate epithelial cell turnover.

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