Screening and Purification of Metal Chelating Peptides in Hydrolysates for their Antioxidant Properties.

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Screening and Purification of Metal Chelating Peptides in Hydrolysates for their Antioxidant Properties.

Laetitia Canabady-Rochelle, Cédric Paris, Katalin Selmeczi, Caroline Gaucher, Igor Clarot, Pierre Leroy, Halima Alem, Raphaël Schneider, Patrick

Chaimbault, Laurence Muhr, et al.

To cite this version:

Laetitia Canabady-Rochelle, Cédric Paris, Katalin Selmeczi, Caroline Gaucher, Igor Clarot, et al.. Screening and Purification of Metal Chelating Peptides in Hydrolysates for their Antioxidant Proper-ties.. ISANH 2016, 2016, Paris, France. �hal-01391695�

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Screening and Purification of Metal Chelating Peptides in Hydrolysates for their

Antioxidant Properties

Laetitia Canabady-Rochelle

1

_{, Cédric Paris}

2

_{, Katalin Selmeczi}

3

_{, Caroline Gaucher}

4

_{, Igor Clarot}

4

_{, Pierre Leroy}

4

_{, Halima Alem}

5

_,

Raphaël Schneider

1

_{, Patrick Chaimbault}

3

_{, Laurence Muhr}

1

_{and Sandrine Boschi-Muller}

6

Université de Lorraine (France): 1_{Laboratoire Réactions et Génie des Procédés (UMR 7274 CNRS-UL),}2_{Laboratoire d’Ingénierie des Biomolécules (EA 4367),}3_{Laboratoire Structure et}

Réactivité des Systèmes Moléculaires Complexes (UMR 7565 CNRS-UL), 4_{EA 3452 Cibles Thérapeutiques Formulation et expertise préclinique du médicament,}5_{Institut Jean Lamour}

(UMR 7198 CNRS-UL), 6_{Laboratoire Ingénierie Moléculaire et Physiopathologie Articulaire (UMR 7365 CNRS-UL).}

laetitia.canabady-rochelle@univ-lorraine.fr

Conclusion and Perspectives

Societal context

Introduction

Reactive Oxygen Species (ROS) induced oxidation

Ageing and various pathologies in human related to oxidative

stress, in particular inflammatory bowel diseases with iron

anemia complication

Degradation of consumption products (food, cosmetic matrix)

Living organisms developped their own antioxidant defenses

against these ROS

Imbalance

Oxidative stress

Importance to discover natural antioxidants and evaluation of

their bioactivity

An interdisciplinary approach

Some results

SPR using a M2+_-chip:

-  a miniaturized approach of IMAC-type affinity chromatography

-  screening of the most interesting sources of metal-chelating peptides.

Positive correlations evidenced between SPR screening and metal chelation tests using UV-Vis spectrometry on five sources of peptides.

The affinity parameters are determined by SPR on some synthetic model peptides and then used to predict the separation of peptides on IMAC chromatographic column.

Novel chromatography-MS method currently developed:

-  Screening of metal-chelating peptides sequences in a given complex mixture -  Methodology successfully set-up on a mixture of synthetic peptides

-  Further tests on real hydrolysates

Identified peptides currently assessed for their antioxidant activity by chemical metal chelation and by in cellulo tests.

Potential health application

-  Treatment of ferriprive anemia related to MICI, or inversely to hematochromatosis

-  Anchoring of these chelating peptides on nanoparticles for bioimaging or therapeutic applications.

Metal-chelating peptides obtained after protein hydrolysis

-  Antioxidant properties related to the inhibition of Fenton reaction, responsible

of ROS production

-  Used in pharmaceutics, foods and cosmetic

Novel strategy for the screening and purification of the peptides to target

their antioxidant properties. Novel approach developed in order to save time.

Currently developed methods for the

screening and

separation of metal-chelating peptides

by Surface Plasmon Resonance (SPR)

by mass spectrometry (MS) :

identification of the peptide(s) of

interest present in the complex

mixture

Determination of metal

_{chelation capacity}

using UV-visible

spectrophotometry

Si g n al in te n si ty /c p s m/z Si g n al in te n si ty /c p s Peptide Peptide-Fe2+ (A) (B)

Integration of these SPR data for

separation prediction in Immobilized

Metal ion Affinity Chromatography

(IMAC)

Pharmacology study of metal

chelating peptides in cellulo:

determination of their intestinal

permeability

Understanding of peptide-metal

complexation, SPR and MS results

Porous&membrane& Caco.2&intes2nal& cells& Free&pep2des&or&metal.pep2de&& complexes&& Analysis(of(absorbed(pep0des( Quan2ﬁca2on&of&free&amine&using&OPA&test& Capillary&electrophoresis&&&&&&&&&pep2des&separa2on&and&iden2ﬁca2on& Si g n al in te n si ty /c p s m/z Si g n al in te n si ty /c p s Peptide Peptide-Fe2+ Metal chelating peptide

Health

applications

Coordination chemistry

Nanoparticules for

bioimaging

0 50 100 150 200 250 300 350 0 10 20 30 40 50 R e s p o n s e (R .U .)

Concentration (mM eq. Glycine)

H1510 H1512 H4601N H5603N UP Soy H1510 Exp. Points H1512 Exp. Points H4601N Exp. Points UP Soy Exp. Points H5603 Exp. Points (A) Screening of complex mixture of peptides by SPR Identification by MS of the peptide(s) of

interest present in the complex mixture.

MS (Orbitrap, ESI, Direct infusion)

solution. (A) without

Fe2+_{. (B) with Fe}2+_.

Mass shift of +56 u and isotopic pattern = iron-containing ion.

Processes and

Modelling

17th International conference on Oxidative Stress, Reduction and Redox Homeostasis

Paris, June 13-15, 2016

Empirical approach Fractioning SEPARATION Complex mixture of peptides H₂N HYDROLYSAT INTERMEDIAIRE H₂N COOH COOH H2N PROTEINES VEGETALES COOH HYDROLYSAT FINAL Peptide bioactif intermédiaire Peptide bioactif H₂N HYDROLYSAT INTERMEDIAIRE H₂N COOH COOH H2N PROTEINES VEGETALES COOH HYDROLYSAT FINAL Peptide bioactif intermédiaire Peptide bioactif Antioxidant peptide ANTIOXIDANT TEST MS-MS IDENTIFICATION Antioxidant bioactive peptide

Rapid screening by MS of metal chelating

peptides SEPARATION Single peptide ? Yes No

Screening of metal chelating peptides for their antioxidant properties using SPR

Novel strategy NTA complexing agent Carboxymethylated dextran as matrix Immobilized metal ion M2+ Ni2+

Screening of metal-chelating

peptides

M(II) M(II) M(II)