Ecotoxico-Proteomics for Aquatic Environmental Monitoring: First in

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Ecotoxico-proteomics for aquatic environmental monitoring: first in situ application of a new proteomicsbased multibiomarker assay using caged amphipods Duarte Gouveia, Arnaud Chaumot, Aurore Charnot, Christine Almunia, Adeline François, Lionel Navarro, Jean Armengaud, Arnaud Salvador, and Olivier Geffard Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03736 • Publication Date (Web): 25 Oct 2017 Downloaded from http://pubs.acs.org on October 25, 2017

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Environmental Science & Technology

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Ecotoxico-proteomics for aquatic environmental monitoring: first

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in situ application of a new proteomics-based multibiomarker

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assay using caged amphipods

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Duarte Gouveia1,2, Arnaud Chaumot1, Aurore Charnot3, Christine Almunia2, Adeline

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François1, Lionel Navarro4, Jean Armengaud2, Arnaud Salvador3, Olivier Geffard1 *

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1

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2

Irstea, UR MALY, Laboratoire d’écotoxicologie, F-69625 Villeurbanne, France CEA-Marcoule, DRF/Joliot/DMTS/SPI/Li2D, Laboratory « Innovative technologies for

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Detection and Diagnostics », Bagnols-Sur-Cèze, F-30207, France

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3

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Analytiques, UMR 5280, 5 rue de la Doua, F-69100 VILLEURBANNE, France.

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Univ Lyon, Université Claude Bernard Lyon 1, CNRS, ENS de Lyon, Institut des Sciences

Agence De L’Eau Rhone Mediterranée Corse, F-69363 Lyon, France

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* Corresponding Author: Olivier Geffard

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Irstea, UR MALY, Laboratoire d’écotoxicologie, Centre de Lyon-Villeurbannne, 5 rue de la

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Doua, CS 20244, F-69625 Villeurbanne Cedex, France

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Tel: +33 (0)4 72 20 87 85

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Email: [email protected]

1 ACS Paragon Plus Environment


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Abstract

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As a proof of principle, a Selected Reaction Monitoring (SRM) mass spectrometry-based

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methodology was applied to the simultaneous quantification of dozens of protein

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biomarkers in caged amphipods (Gammarus fossarum). We evaluated the suitability of the

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methodology to assess complex field contaminations through its application in the

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framework of a regional river monitoring network. Thanks to the high throughput

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acquisition of biomarker levels in G. fossarum exposed in four reference and thirteen

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contaminated sites, we analyzed the individual responses of 38 peptides reporting for 25

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proteins of interest in 170 organisms. Responses obtained in contaminated sites included

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inductions of vitellogenin-like proteins in male organisms, inductions of Na+K+/ATPases,

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and strong inhibitions of molt-related proteins such as chitinase and JHE-

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carboxylesterase. Proteins from detoxification and immunity processes were also found

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modulated in abundance. Summarizing, the results presented here show that the SRM

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strategy developed for multi-biomarker measurement paves a very promising way to

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define multiple indicators of the health status of sentinel organisms for environmental

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hazard assessment.

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Keywords

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Biomarkers, biomonitoring, ecotoxicology, Gammarus fossarum, mass spectrometry,

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multiplex, targeted proteomics


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Introduction

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In amphipod crustaceans, some well-established biomarkers were developed in the last

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decades, such as the protein biomarkers acetylcholinesterase (AChE)

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detoxification/antioxidant enzymes glutathione S-transferase (GST), catalase (CAT) and

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superoxide dismutase (SOD) 2, or digestive enzymes3. However, their use in routine

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biomonitoring is still limited by several technical factors like the lack of direct

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quantification methods in non-model sentinel species. The fact that each assay is specific

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for one biomarker of interest, mainly through the measurement of enzymatic activities,

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multiplies laboratory work, cost, and time necessary for the analysis of a large number of

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samples.

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Hence, the development of multi-biomarker strategies that allow the simultaneous

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monitoring of a wide range of biological responses is required for improving

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environmental hazard assessment. In the sentinel amphipod Gammarus fossarum, a

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targeted proteomics method was recently developed for the simultaneous quantification

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of 40 proteins, using Selected Reaction Monitoring (SRM) mass spectrometry 6. This

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methodology has been used to validate candidate proteins with key physiological roles in

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order to propose them as biomarkers of toxicity 7. These studies highlighted the great

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potential of this methodology in ecotoxicology, due to its capacity to monitor

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simultaneously an ensemble of protein biomarkers from one single organism, and to its

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reduced technical variability, higher sensitivity, and lower detection limits when

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compared to shotgun methods 8.

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However, it is now mandatory to make proof of principle of the use of these techniques in

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field applications. One drawback is the lack of control of biological factors of sentinel

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organisms (genetic background, age, sex, reproductive status), which introduce variability

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in proteomic biomarker responses in the field, especially when comparing different

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populations 9, 10. For instance, while gender has no influence in some biomarker responses, 3 ACS Paragon Plus Environment

1,

the


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such as the AChE activity in gammarids 1, it has been reported as a source of confusion in

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several studies. For example, Sornom et al.

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Gammarus roeseli, gender influences the organisms responses to a salinity and a heavy

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metal stress, respectively. To overcome these difficulties of confounding biological factors,

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a robust active biomonitoring strategy based on caged organisms from the same reference

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population, was developed with G. fossarum and applied to several in situ studies 11-14. G.

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fossarum is an ecologically relevant test species commonly used for freshwater

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monitoring. They are widespread in rivers around Europe, occurring often in high

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densities, and are easily identifiable to the species level. Data are available for a wide

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range of stressors and lethal and sublethal responses for this genus. Moreover, a

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reproduction toxicity test is available for G. fossarum 15.

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The objective of the present study was to assess the suitability of the SRM mass-

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spectrometry-based multibiomarker methodology developed in G. fossarum for field

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studies. For this, protein biomarker quantitation was carried out during a caging

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deployment operated in South-Eastern France throughout the regional river monitoring

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network from the “Rhône Méditerranée Corse” (RMC) French Water Agency. We analyzed

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caged male and female organisms in 4 reference sites and 13 sites subjected to chemical

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contamination, previously monitored and prioritized by the French Water Agency. After

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exposure, we applied the SRM strategy to analyze 38 peptides that report for 25 protein

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biomarkers of interest (involved in reproduction, immunity, homeostasis, detoxification,

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and defense mechanisms). Firstly, our aim was to evaluate the robustness of the

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methodology and its capacity to detect and describe the possible response patterns of

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biomarkers in male and female organisms, when facing a complex and realistic field

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exposure. Thirdly, we aimed at identifying proteins whose modulations were

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physiologically relevant, in order to propose robust biomarkers of toxic exposure in G.

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fossarum.

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and Gismondi et al.


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observed that in

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Materials and Methods

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Sampling, selection and field exposure of caged organisms

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Gammarids were collected from the Bourbre River in France from a source population

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commonly used in our laboratory

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previously described

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quickly transported to the laboratory. In the laboratory, organisms were kept in 30 L tanks

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continuously supplied with drilled groundwater and under constant aeration for at least

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10 days. The drilled groundwater water from the tanks was adjusted to a conductivity of

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600±20 μS/cm, pH of 7.4±0.2, with a flow rate of 2.25 L/h. A 16/8h light/dark

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photoperiod was maintained and the temperature was kept at 12±1°C. Organisms were

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fed ad libitum with alder leaves (Alnus glutinosa), previously conditioned for 6±1 days

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using the same drilled groundwater water.

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Male and female gammarids in amplexus (female at final D2 molting stage) were caged

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and fed ad libitum with alder leaves during exposure. Organisms were placed in punctured

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polypropylene cylinders to allow free circulation of water. One polypropylene cylinder

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containing 10 couples was placed in each site (a total of 10 males and 10 females in a

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common cylinder per site). Each exposure was conducted from the female final D2 molting

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stage (day 0) until they reach the C2/D1 stage. Since the gammarid molting cycle is

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temperature-dependent, the duration of exposure was different among sites (Table 1).

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This ensures that the female organisms were exposed during new starting reproductive

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cycle, up to a determined stage of follicular maturation and marsupial embryonic

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development (see Geffard et al. 2010 for details

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reproductive status (no copulation after the initial fertilization at the beginning of the

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exposure). At the end of exposure, organisms were counted (for survival rate assessment),

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weighed, and directly frozen in liquid nitrogen and stored at -80°C until further analysis.

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11, 12, 14, 16

and acclimatized to laboratory conditions as

G. fossarum were collected by kick sampling using a net, and

15),

and that males are in a common



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Study sites

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The 17 study sites belong to two river monitoring networks implemented by the French

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Water Agency Rhône-Méditerranée-Corse (South Eastern France), in an attempt to meet

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the Water Framework Directive (WFD) requirements. The surveillance-monitoring

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network aims to allow the assessment of long-term changes in natural conditions, and to

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provide an assessment of the overall surface water status within each catchment and sub-

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catchment of the river basin district. This network is complemented by the operational

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monitoring network, which aims to establish the status of water bodies identified as being

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at risk of failing to meet the WFD environmental objectives and their evolution in response

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to remediation actions. Seventeen sites, which have been intensively monitored since

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more than 10 years by the Agency, were selected among these observation networks on a

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large geographical scale (Figure S1). Hence, the study considered four reference sites

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versus thirteen contaminated sites. These sites are in fact situated on water bodies, for

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which the Agency previously assessed the risk of failing to achieve good chemical and

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ecological quality in 2021, due to specific adverse anthropic influences (Table 1). The risk

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related to micro-pollutant contamination was estimated through the expert system

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developed by the regulatory Water Agency, which consists in a standardized procedure

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allowing the integration of heterogeneous quantitative data from different sources

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(experimental observations, model outputs) and expert judgment. More precisely, this

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expert system mainly considers data from multi-year chemical monitoring of toxic

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substances in water and sediments (including WFD priority substances), data from the

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detected and predicted levels of contaminants in connected industrial and wastewater

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treatment plant effluents, and records from land cover analysis notably to quantify

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pesticide pressure.

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Biomarker peptides/proteins used in the multiplexed SRM assay

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Quantified proteins are listed in Table S1. Proteins and peptides were selected

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based on their putative physiological role, given by their bioinformatic functional

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annotations provided in the GFOSS database (see Trapp et al. 2014 {Trapp, 2014 #198}).

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The protein list sought to cover different important physiological functions potentially

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subject to disruption by exposure to contaminants: sex-specific and reproduction-related

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proteins, and proteins whose annotations suggested roles in immunity, homeostasis,

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detoxification and defence mechanisms. The reporter peptides used for each protein were

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validated in previous studies 6, 7.

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Each peptide is assigned to a specific protein ID (some peptides belong to the same

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protein ID), and each protein ID corresponds to an assembled contig (=one putative

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protein) from the RNAseq GFOSS database

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common functional annotation. Because the GFOSS database is a de novo assembled

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RNAseq, we cannot ascertain whether distinct protein IDs with the same annotation

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correspond to duplicated paralog genes, to alternatively spliced isoforms of a unique gene,

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or to redundant contigs in the de novo assembled RNAseq. The targeted functions were

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considered as crucial for the reproduction, homeostasis, and defense mechanisms of the

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organisms. Therefore, the chosen biomarkers could give important insights into toxic

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disruption of these key physiological processes.

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Protein analysis by LC-MS/MS

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Five male and five female organisms were analyzed per site. The procedures used for total

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protein extraction and digestion were the same as in our previous studies 6. Mass

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spectrometry analysis was performed using the targeted mass spectrometry mode SRM.

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This technique uses a two-stage mass filtering to analyze previous selected pairs of

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precursor/product ions in a triple quadrupole mass spectrometer. Compared to the

18.

Different protein IDs share sometimes a



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untargeted full scan MS/MS analyses, the SRM approach is much more sensitive (in the

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order of 100 fold) and selective regarding quantitative analyses. The scheduled SRM

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algorithm from Analyst 1.5 software was used for creating short time windows around the

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peptides expected retention time, therefore improving the maximum number of

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transitions and diminishing analysis time. Three transitions per peptide were monitored.

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Heavy labeled isotopologs of target peptides of known concentration were spiked into the

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samples, and concomitantly monitored to ensure precise and accurate measurements of

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endogenous peptide concentrations.

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LC–MS/MS analysis was performed with an HP1200 series HPLC device (Agilent

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Technologies, Waldbronn, Germany) coupled to a QTRAP® 5500 LC/MS/MS System

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hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems/ MDS

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Analytical Technologies, Foster City, CA, USA) equipped with a Turbo VTM ion source. The

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LC separation of the 20 µL injected sample was carried out on an Xbridge C18 column

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(100mm×2.1mm, particle size 3.5 µm) from Waters (Milford, MA, USA). Details concerning

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sample preparation, chromatographic separation of peptides and mass spectrometric

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analysis can be found in Supporting Information.

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Quantification and statistical analysis

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Peptide absolute concentrations were calculated using the endogenous/labelled peptide

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peak area ratio (peak analysis with Skyline v3.1, MacCoss Lab Software, USA), since the

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amount of labelled peptide was known (1000 ng/mL).

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Results are presented either as heatmaps (heatmap.2 function in R software), or as min

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max boxplots of five biological replicate samples. Data are reported in pmol of peptide per

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milligram of organism wet-weight. All graphs and statistical analysis for testing significant

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differences in peptide concentrations between conditions were performed with the

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GraphPad Prism Version 7.02 software. Non-parametric Kruskal-Wallis tests with Dunn’s


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corrections for multiple comparisons analysis were used for comparison between

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reference and contaminated sites. Significant differences were accepted at p-values

Ecotoxico-Proteomics for Aquatic Environmental Monitoring: First in

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