Dataset Information

Phosphoproteomic analysis in brain of fathead minnows exposed to ethinyl estradiol and levonorgestrel

ABSTRACT: Adverse outcome pathways (AOPs) are conceptual frameworks that organize and link contaminant-induced molecular changes to adverse biological responses at the individual and population level. The development of AOPs is a priority area of research in ecotoxicology as a means to leverage molecular and high content mechanistic information obtained through top-down ‘omic approaches in regulatory decision-making. Current AOPs for hormonally active agents (HAAs) focus on nuclear receptor-mediated effects despite the overwhelming evidence that HAAs also activate membrane receptors. Activation of membrane receptors triggers propagation of non-genomic signaling cascades often transduced by protein phosphorylation leading to phenotypic changes. To promote investigations of rapid non-genomic signaling mechanisms of environmental contaminants in aquatic species for top-down AOP development, we developed a phosphoproteomic pipeline using label-free LC-MS/MS. We used our optimized pipeline to identify proteins differentially phosphorylated in the brain of fathead minnows (Pimephales promelas) aqueously exposed for 30 minutes to two HAAs, 17α-ethinylestradiol (EE2), a strong estrogenic substance, and levonorgestrel (LNG), a progestin, both components of the birth control pill. Results revealed differential phosphorylation of proteins involved in neuronal processes such as nervous system development, synaptic transmission, and neuroprotection by EE2 while LNG induced differential phosphorylation of proteins involved in axon cargo transport and calcium ion homeostasis. EE2 and LNG caused similar enrichment of cell processes including synaptic plasticity and neurogenesis. This study is the first to identify molecular changes in vivo in fish after short-term exposure and highlights membrane receptors and transduction of rapid, non-genomic signaling mechanisms as targets of HAAs in addition to the well-established nuclear receptor-mediated pathways.

INSTRUMENT(S): LTQ Orbitrap, Q Exactive Plus

ORGANISM(S): Pimephales Promelas

TISSUE(S): Brain

DISEASE(S): Disease Free

SUBMITTER: Ley Smith

LAB HEAD: Tara Sabo-Attwood

PROVIDER: PXD007714 | Pride | 2018-07-11

REPOSITORIES: pride

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Early phosphoproteomic changes for adverse outcome pathway development in the fathead minnow (Pimephales promelas) brain.

Smith L C LC Lavelle C M CM Silva-Sanchez C C Denslow N D ND Sabo-Attwood T T

Scientific reports 20180705 1

Adverse outcome pathways (AOPs) are conceptual frameworks that organize and link contaminant-induced mechanistic molecular changes to adverse biological responses at the individual and population level. AOPs leverage molecular and high content mechanistic information for regulatory decision-making, but most current AOPs for hormonally active agents (HAAs) focus on nuclear receptor-mediated effects only despite the overwhelming evidence that HAAs also activate membrane receptors. Activation of me ...[more]

PMID: 29977039

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Project description:The experiment consists of M.hyp 232 cultures digests analyzed on two mass specs, LTQ Velos Pro and LTQ FT Ultra LTQ Velos Pro: Six cell culture replicates of M.hyo 232 were hydrophobically separated using tree detergents: Digitonin, Tween, and SDS. Each francion, including the insoluble pellet, was trypsin digested and then cleaned using an SCX trap follwed by a RP trap to remove detergents. Each fraction was sepated using a Dionex U3000 splitless nanoflow system operating at 333 nl per minute using a gradient of 2% ACN to 50% ACN in 4 hours. Eluate was analyzed using an LTQ Velos Pro mass spectrometer with 20 MS/MS scans of the 20 most intense peaks from each MS scan. Dynamic exclusion was enable for 3 minutes for each m/z with a repeat count of 1. LTQ FT Ultra: Two cell pellets for high resolution analysis were lysed and trypsin digested. Digested peptides were dried, resuspended in 20 mM KH2PO4, 20% ACN, pH 3 (Buffer A) in 2.5 µL and transferred to low retention vials in preparation for separation using an Ultimate 3000 configured for 2D-LC. Each sample was loaded at 15 µl/min onto an SCX microtrap for the first dimension of separation, involving SCX steps of Buffer A + 0, 5, 10, 15, 20, 25, 30, 40, 50, 100, 250, 500, and 1000 mM KCl. For the second dimension of separation, each eluted salt step was desalted with an inline peptide microtrap with 2% ACN, 0.1% FA at 5 µl/min. Once desalted, the microtrap was switched into line with a fritless nano column (75µm x ~10cm) containing C18 media (5µ, 200 Å Magic, Michrom). Peptides were eluted using a gradient of 2% to 36% ACN, 0.1% FA at 350 nl/min over 60 min and electrospray ionized for analysis using an LTQ FT Ultra mass spectrometer. A survey scan m/z 350-1750 was acquired in the FT ICR cell (Resolution = 100,000 at m/z 400, with an accumulation target value of 1,000,000 ions). Up to the 6 most abundant ions (>3,000 counts) with charge states > +2 were sequentially isolated and fragmented within the linear ion trap using collisionally induced dissociation with an activation q = 0.25 and activation time of 30 ms at a target value of 30,000 ions. M/z ratios selected for MS/ MS were dynamically excluded for 30 seconds. Analysis: X!tandem searches were performed using the Mycoplasma hyopneumoniae strain 232 reference protein set from NCBI. The only difference between the searches for the LTQ Velos and LTQ FT was the precursor mass tolenance being set to +-1500ppm and +-24ppm respectively. Decoy searches were performed and the data filtered at e-value <= 0.01 with single peptide proteins discarded. These results are included in the submission as two tab-delimited text files.

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