Dataset Information

Grayling Pleiotropy on plastic adaptive protein expression change

ABSTRACT: We studied a Norwegian metapopulation of grayling, a spring-spawning freshwater salmonid fish with high homing propensity that has undergone contemporary evolution of early life-history in response to temperature. Samples derived from four spawning sites in four grayling sub-populations in Lake Lesjaskogsvatnet (62 14'N 8 25'E) in southern Norway. Adult grayling were captured at the four spawning sites, stripped of gametes and fertilized eggs were reared in two temperature treatments in common garden conditions as to represent lower and upper temperatures experienced by developing grayling larvae in nature. Proteins were isolated using a standard sodium dodecyl sulphate (SDS)-based extraction method. For trypsin digestion and peptide-level iTRAQ labeling, samples were peptide-labeled with isobaric tags by iTRAQ reagents (4-plex, Applied Biosystems) following the manufacturer's instructions. To clean and fractionate each sample we employed an isotope coded affinity tag procedure (ICAT) using the ICAT Cation Exchange Buffer Pack - Cation Exchange Cartridge system (Applied Biosystems). Four peptide fractions were collected using four concentrations of the elution buffer (50 mM, 100 mM, 150 mM, and 350 mM KH2PO4). Peptides were desalted using C18 macrospin columns (The Nest Group, Southborough, MA). For LC-MS/MS, peptides were separated by reversed-phase chromatography using an Easy-nLC II nanoflow system connected to an Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Peptides were separated at a flow rate of 300 nl/min with 102 min gradients as follows: initially 2 % B to 25% B (60 min), 40% B (90 min), and 100% B (92-102 min). Protein identification and quantification were done using the ProteinPilotTM v.4 program (Applied Biosystems). In the parameters we selected the Paragon method, iodoacetamide for cys alkylation, trypsin for digestion, thorough ID for search effort, biological modifications and amino acid substitutions for ID focus, and bias correction in quantification. As a search database we used all Atlantic salmon (Salmo salar) submitted in the UniProt database (www.uniprot.org) up to March 2012. To evaluate the accuracy of the quantification method, we also did a pilot experiment. We spiked the six-protein mix provided with the iTRAQ kit in ratios 1:3:10 in samples that contained an equal amount of protein extract from a grayling embryo. Then we labeled each sample with a different isobaric tag (114:115:116, respectively). The aim was to employ the proposed methodology to quantify a subset of proteins that change across samples in a cloud of background proteins that remain unchanged and these data are also provided in the dataset.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Thymallus Thymallus (grayling) (salmo Thymallus)

SUBMITTER: Spiros Papakostas

PROVIDER: PXD000368 | Pride | 2014-05-28

REPOSITORIES: pride

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Dataset's files

Source:

			Action	DRS
	0_Explanation_of_Files.txt	Txt
	2_Database_Salmosalar.fasta	Fasta
	2_Database_SixProtein.fasta	Fasta
	3_Reaction1_ProteinPilot.xml	Xml
	3_Reaction2_ProteinPilot.xml	Xml

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Publications

Gene pleiotropy constrains gene expression changes in fish adapted to different thermal conditions.

Papakostas Spiros S Vøllestad L Asbjørn LA Bruneaux Matthieu M Aykanat Tutku T Vanoverbeke Joost J Ning Mei M Primmer Craig R CR Leder Erica H EH

Nature communications 20140603

Understanding the factors that shape the evolution of gene expression is a central goal in biology, but the molecular mechanisms behind this remain controversial. A related major goal is ascertaining how such factors may affect the adaptive potential of a species or population. Here we demonstrate that temperature-driven gene expression changes in fish adapted to differing thermal environments are constrained by the level of gene pleiotropy estimated by either the number of protein interactions ...[more]

PMID: 24892934

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