Proteome analysis of male gonads in Gammarus fossarum exposed to Pyriproxyfen: mining for endocrine disruption biomarkers

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Proteome analysis of male gonads in Gammarus

fossarum exposed to Pyriproxyfen: mining for endocrine disruption biomarkers

D. Gouveia, Judith Trapp, K. Abbaci, L. Guillet Revol, Geffard Olivier, Arnaud Chaumot, C. Almunia, J. Armengaud

To cite this version:

D. Gouveia, Judith Trapp, K. Abbaci, L. Guillet Revol, Geffard Olivier, et al.. Proteome analysis of male gonads in Gammarus fossarum exposed to Pyriproxyfen: mining for endocrine disruption biomarkers. SETAC Europe 27th Annual Meeting, May 2017, Brussels, Belgium. pp.1, 2017. �hal-02606961�

Proteome analysis of male gonads in Gammarus fossarum exposed

to Pyriproxyfen: mining for endocrine disruption biomarkers

Duarte Gouveia

1,2

_{, Judith Trapp}

1,2

_{, Khédidja Abbaci}

1

_{, Laurent Guillet-Revol}

1

_{, Olivier Geffard}

1

_{, Arnaud Chaumot}

1

_,

Christine Almunia

2

_{, Jean Armengaud}

2

1

_{Irstea, Unité de Recherche MALY, Laboratoire d’écotoxicologie, F-69626 Villeurbanne, France}

2

_{CEA-Marcoule, DRF-Laboratoire “Innovative technologies for Detection and Diagnostics”, Bagnols-sur-Ceze, F-30207, France}

E-mail contact: duarte.gouveia@irstea.fr



Proteomics is becoming increasingly popular in Ecotoxicology for the discovery of new candidate biomarkers of environmental contamination



Recent technological advances in high-throughput “omics” methodologies, in which thousands of genes, proteins or metabolites can be assessed

simultaneously, provide invaluable molecular information



In order to unveil the novel perspectives offered by the latest generation of mass analyzers, we analyzed the performances of the Q-Exactive HF tandem

mass spectrometer equipped with an ultra-high field Orbitrap analyzer

OBJECTIVE: The aim of this study was to reconsider the discovery of new candidate biomarkers of exposure to an endocrine disruptor in male Gammarus

fossarum by shotgun proteomics, in the light of the latest generation of tandem mass spectrometer

Methodology

Results

0,5 and 50 µg/L

Pyriproxyfen Exposure

during two consecutive

spermatogenesis

Mass Range

Dynamic

Range Mass Accuracy Polarity Switching Sensitivity Resolution 50–6000 m/z >5000:1 Internal: <1 ppm RMS External: <3 ppm RMS

One full cycle in <1 sec (at a resolution setting of 60,000) Full MS: 500 fg buspirone on column S/N 100:1 SIM: 30 fg buspirone on column S/N 100:1 Up to 240,000 (FWHM) at m/z 200

Gonad dissection

Trypsin digestion

Protein extract

Protein purification

SDS-PAGE 1D

Protein tryptic in

gel digestion

LC-MS/MS analysis

Bioinformatic analysis



Protein identification



Spectral count

quantitation



Patternlab analysis for

modulated proteins

Conclusion

P

yriproxyfen modulated proteins



A total of 53 significantly modulated proteins in both

conditions (blue dots)

Table 1. Summary and statistics of G. fossarum proteome datasets.

Control PYR 0.5 µg/L PYR 50 µg/L

MS/MS spectra Mean 55769 56442 55333 SD 3899 9619 6647 CV 7% 17% 12% Assigned spectra Mean 12265 12176 12298 SD 1243 2261 2116 CV 10% 19% 17% Unique peptides Mean 5453 4998 5328 SD 375 855 1200 CV 7% 17% 23% Identified proteins Mean 1515 1309 1305 SD 89 179 275 CV 6% 14% 21%

Introduction

Inducted proteins Inhibited proteins

Acknowledgments: This work was financed by the Commissariat à l'énergie atomique et aux énergies alternatives de Marcoule, the IRSTEA Lyon-Villeurbanne and ANR Proteogam project

P

rotein identification



PatternLab analysis

L-stringency = 0.6

P value < 0.05

FDR < 0.05

Fold change > 2

Seen in 4/5 replicate samples



Spectra were assigned to peptide sequences from GFOSS

proteogenomics database derived from RNAseq

(Trapp et.al, Mol Cell Proteomics. 2014 Dec; 13(12): 3612–3625.)



22% total spectra assigned



The Q Exactive HF allows recording five fold more peptides

and proteins than the LTQ Orbitrap XL (data not shown)



Faster scan rate (20Hz) of the ultra-high-field

Orbitrap analyzer



Incorporation of a quadrupole in the instrument

configuration

4031 identified proteins (with at least 2 peptides)

2047 validated proteins with Evalue < -10

P. Carvalho, “Integrated analysis of shotgun proteomic data with PatternLab for proteomics 4.0”, Nature Protocols (11), 102-117 (2016)



Next-generation instruments based on the ultra-high-field Orbitrap analyzer outperform previous generations in achieving more quickly the same comparative proteomics overview



Due to its faster scan rate, the quadrupole filtering properties and the higher resolution on MS/MS secondary ions, the Q Exactive HF instrument is able to record almost 5 fold more

MS/MS spectra and 4 times more proteins than the LTQ Orbitrap XL, and moreover it increases the intrinsic quality of these spectra



However, no reproduction-related proteins were identified as being modulated by pyriproxyfen (that met both fold change and statistical criteria)



By mining through the background noise of the results, physiologically-pertinent candidates that didn’t meet all statistical criteria were found with strong fold changes in comparison to

control samples, and can be further analyzed

NCBInr first homolog protein

Patternlab

Classification Fold Change pValue evalue Accession NCBInr Functional annotation Organism

Blue 29,00 0,03422 2E-20 CAI78901.1 hemocyanin subunit 1 Gammarus roeselii

Green 16,00 0,12322 4,3E-39 XP_018024698.1 PREDICTED: hemolymph clottable protein-like isoform X1 Hyalella azteca

Green 11,00 0,19586 1,9E-67 XP_018023978.1 PREDICTED: hemocyanin B chain-like Hyalella azteca

Orange 10,00 0,04257 4,1E-55 XP_018023786.1 PREDICTED: glutathione S-transferase 1-like Hyalella azteca

Orange -3,67 0,00361 8,3E-69 XP_018016475.1 PREDICTED: catalase-like Hyalella azteca

Green -7,00 0,07996 1,9E-28 AAX98287.1 hepatopancreas trypsin, partial Astacus leptodactylus

Green -7,50 0,07811 1,1E-44 XP_018016810.1 PREDICTED: endoglucanase A-like Hyalella azteca

Green -8,00 0,05138 1,2E-51 XP_018028141.1 PREDICTED: prostaglandin reductase 1-like Hyalella azteca

Green -11,00 0,05754 1,5E-84 XP_018015212.1 PREDICTED: juvenile hormone epoxide hydrolase 1-like Hyalella azteca

Green -13,50 0,08262 1,6E-49 XP_018016813.1 PREDICTED: endoglucanase E-4-like Hyalella azteca

Green -23,00 0,15785 5,7E-24 XP_014260234.1 PREDICTED: glutathione S-transferase 1, isoform D-like Cimex lectularius

Green -18,00 0,08898 4,2E-39 XP_018015052.1 PREDICTED: cuticle protein AM1159-like Hyalella azteca

Green -12,00 0,08815 3,9E-136 XP_018024668.1 PREDICTED: endochitinase-like Hyalella azteca

Green -12,00 0,16996 5,4E-38 XP_018027468.1 PREDICTED: glutathione S-transferase 2-like Hyalella azteca

Blue -8,57 0,00233 4,6E-38 XP_014260234.1 PREDICTED: glutathione S-transferase 1, isoform D-like Cimex lectularius

Orange 8,00 0,04153 8,8E-42 XP_018018288.1 PREDICTED: prostaglandin E synthase 3-like Hyalella azteca

Blue 16 0,01230 2E-20 CAI78901.1 hemocyanin subunit 1 Gammarus roeselii

P

YR 0.5

µg

/L

P

YR

50

µg

/L



By only applying fold change criteria, other physiological relevant proteins were found modulated:

• Proteins mainly connected

to housekepping functions

• Not relevant from a

physiological point of view

to document reproductive

disorders