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      Plasma proteome profiling of freshwater and seawater life stages of rainbow trout ( Oncorhynchus mykiss)

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          Abstract

          The sea-run phenotype of rainbow trout ( Oncorhynchus mykiss), like other anadromous salmonids, present a juvenile stage fully adapted to life in freshwater known as parr. Development in freshwater is followed by the smolt stage, where preadaptations needed for seawater life are developed making fish ready to migrate to the ocean, after which event they become post-smolts. While these three life stages have been studied using a variety of approaches, proteomics has never been used for such purpose. The present study characterised the blood plasma proteome of parr, smolt and post-smolt rainbow trout using a gel electrophoresis liquid chromatography tandem mass spectrometry approach alone or in combination with low-abundant protein enrichment technology (combinatorial peptide ligand library). In total, 1,822 proteins were quantified, 17.95% of them being detected only in plasma post enrichment. Across all life stages, the most abundant proteins were ankyrin-2, DNA primase large subunit, actin, serum albumin, apolipoproteins, hemoglobin subunits, hemopexin-like proteins and complement C3. When comparing the different life stages, 17 proteins involved in mechanisms to cope with hyperosmotic stress and retinal changes, as well as the downregulation of nonessential processes in smolts, were significantly different between parr and smolt samples. On the other hand, 11 proteins related to increased growth in post-smolts, and also related to coping with hyperosmotic stress and to retinal changes, were significantly different between smolt and post-smolt samples. Overall, this study presents a series of proteins with the potential to complement current seawater-readiness assessment tests in rainbow trout, which can be measured non-lethally in an easily accessible biofluid. Furthermore, this study represents a first in-depth characterisation of the rainbow trout blood plasma proteome, having considered three life stages of the fish and used both fractionation alone or in combination with enrichment methods to increase protein detection.

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          Database searching and accounting of multiplexed precursor and product ion spectra from the data independent analysis of simple and complex peptide mixtures.

          A novel database search algorithm is presented for the qualitative identification of proteins over a wide dynamic range, both in simple and complex biological samples. The algorithm has been designed for the analysis of data originating from data independent acquisitions, whereby multiple precursor ions are fragmented simultaneously. Measurements used by the algorithm include retention time, ion intensities, charge state, and accurate masses on both precursor and product ions from LC-MS data. The search algorithm uses an iterative process whereby each iteration incrementally increases the selectivity, specificity, and sensitivity of the overall strategy. Increased specificity is obtained by utilizing a subset database search approach, whereby for each subsequent stage of the search, only those peptides from securely identified proteins are queried. Tentative peptide and protein identifications are ranked and scored by their relative correlation to a number of models of known and empirically derived physicochemical attributes of proteins and peptides. In addition, the algorithm utilizes decoy database techniques for automatically determining the false positive identification rates. The search algorithm has been tested by comparing the search results from a four-protein mixture, the same four-protein mixture spiked into a complex biological background, and a variety of other "system" type protein digest mixtures. The method was validated independently by data dependent methods, while concurrently relying on replication and selectivity. Comparisons were also performed with other commercially and publicly available peptide fragmentation search algorithms. The presented results demonstrate the ability to correctly identify peptides and proteins from data independent acquisition strategies with high sensitivity and specificity. They also illustrate a more comprehensive analysis of the samples studied; providing approximately 20% more protein identifications, compared to a more conventional data directed approach using the same identification criteria, with a concurrent increase in both sequence coverage and the number of modified peptides.
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            Methods for Nonlethal Gill Biopsy and Measurement of Na+, K+-ATPase Activity

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              Mass Spectrometry-Based Label-Free Quantitative Proteomics

              In order to study the differential protein expression in complex biological samples, strategies for rapid, highly reproducible and accurate quantification are necessary. Isotope labeling and fluorescent labeling techniques have been widely used in quantitative proteomics research. However, researchers are increasingly turning to label-free shotgun proteomics techniques for faster, cleaner, and simpler results. Mass spectrometry-based label-free quantitative proteomics falls into two general categories. In the first are the measurements of changes in chromatographic ion intensity such as peptide peak areas or peak heights. The second is based on the spectral counting of identified proteins. In this paper, we will discuss the technologies of these label-free quantitative methods, statistics, available computational software, and their applications in complex proteomics studies.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Writing – original draft
                Role: Formal analysisRole: MethodologyRole: ResourcesRole: SoftwareRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: MethodologyRole: SupervisionRole: Writing – review & editing
                Role: Funding acquisitionRole: MethodologyRole: Project administrationRole: ResourcesRole: Writing – review & editing
                Role: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: Funding acquisitionRole: Project administrationRole: Resources
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                3 January 2020
                2020
                : 15
                : 1
                : e0227003
                Affiliations
                [1 ] Institute of Aquaculture, University of Stirling, Stirling, Scotland, United Kingdom
                [2 ] Institute of Health Research and Innovation, Centre for Health Science, University of the Highlands and Islands, Inverness, Scotland, United Kingdom
                [3 ] NORCE AS, Universitetet i Bergen, Bergen, Norway
                [4 ] Lerøy Seafood Group ASA, Universitetet i Bergen, Bergen, Norway
                Northwest Fisheries Science Center, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-6799-3882
                Article
                PONE-D-19-24262
                10.1371/journal.pone.0227003
                6941806
                31899766
                ed0d5d26-f6b2-4414-b1b4-3af3613498a3
                © 2020 Morro et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 30 August 2019
                : 9 December 2019
                Page count
                Figures: 6, Tables: 1, Pages: 23
                Funding
                Funded by: Regionale Foskningsfond Vestalandet project
                Award ID: RFFVest project 248020
                Award Recipient :
                Funded by: CtrlAQUA
                Award ID: SFI project 237856
                Award Recipient :
                Funded by: MASTS
                Award ID: SASG9
                Award Recipient :
                This work was supported by the Regionale Foskningsfond Vestalandet project: Utvikling av en sesonguavhengig protokoll for intensiv produksjon av regnbueørret (O. mykiss) (Development of a season independent protocol for the intensive production of rainbow trout (O. mykiss)) (RFFVest project 248020), the CtrlAQUA SFI, Centre for Closed-Containment Aquaculture programme (SFI project 237856), and by Japan Society for the Promotion of Science Bilateral Joint Research Project (Open Partnership with Norway). This work also received funding from the MASTS pooling initiative (The Marine Alliance for Science and Technology for Scotland; grant SASG9) and their support is gratefully acknowledged. MASTS is funded by the Scottish Funding Council (grant reference HR09011) and contributing institutions.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Proteins
                Plasma Proteins
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Vertebrates
                Fish
                Osteichthyes
                Trout
                Biology and Life Sciences
                Anatomy
                Body Fluids
                Blood
                Blood Plasma
                Medicine and Health Sciences
                Anatomy
                Body Fluids
                Blood
                Blood Plasma
                Biology and Life Sciences
                Physiology
                Body Fluids
                Blood
                Blood Plasma
                Medicine and Health Sciences
                Physiology
                Body Fluids
                Blood
                Blood Plasma
                Ecology and Environmental Sciences
                Aquatic Environments
                Marine Environments
                Sea Water
                Earth Sciences
                Marine and Aquatic Sciences
                Aquatic Environments
                Marine Environments
                Sea Water
                Biology and Life Sciences
                Biochemistry
                Proteins
                Proteomes
                Biology and Life Sciences
                Biochemistry
                Proteins
                Serum Proteins
                Ecology and Environmental Sciences
                Aquatic Environments
                Freshwater Environments
                Fresh Water
                Earth Sciences
                Marine and Aquatic Sciences
                Aquatic Environments
                Freshwater Environments
                Fresh Water
                Biology and Life Sciences
                Biochemistry
                Biomarkers
                Custom metadata
                All relevant data are within the paper and its Supporting Information files. All Raw mass spectrometry files are available from the ProteomeXchange database (accession number(s) PXD016570).

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