Design of microsystems meant for phosphopeptides analysis: toward accurate diagnosis of pathologies

Master's internship, AAP 2017

Team: Nanomedicines for the treatment of severe diseases

Project leader : Talal Mallah

Abstract :

Phosphorylation is a post-translational modification (PTM) playing a critical role in the regulation of many cellular processes including metabolism. However, anomalous proteins’ phosphorylation may be associated to cancers or neurodegenerative diseases [1]. To develop a diagnosis based on phosphorylated biomarkers, innovative methods for enrichment to improve limit of detection as phosphorylated biomarkers are always present in biological fluids at the trace level. Immobilized metal affinity chromatography (IMAC) is one of the most widely applied approach for phosphor-proteins/peptides preconcentration [2]. Among existing sorbents, monoliths have demonstrated great potential for the pretreatment of complex biological samples owing to their large surface area, controlled porosity and ease of in situ preparation through polymerization [3]. Our laboratory has gained long term expertise in the photochemical-assisted synthesis of polymer- or silica-based monoliths within fused silica capillaries, glass [4] and PDMS [5] microchips. Very recently, we have developed a miniaturized analytical tool integrating a methacrylate-based polymer monolith as solid support for the preconcentration of phosphopeptides coupled to “on line” microchip electrophoresis for the separation of the phosphopeptides [6].

This project aims at designing a microsystem incorporating a monolithic sorbent for the IMAC preconcentration of phosphopeptides and their subsequent electrophoretic separation. The project will be divided in three main tasks:

T1: Synthesis of solid supports within microchannels (glass, COC): Porous monoliths will be prepared through free radical photopolymerization via an unprecedented strategy involving the use of the monomer bearing phosphate groups in an ester protected form. This strategy will enable to prevent solubility problems typically observed in the course of the polymerization of hydrophilic monomers, such as phophonate-containing monomers, in the presence of hydrophobic porogen solvents. After post-polymerization hydrolysis of the phosphate ester, monolith decorated with surface phosphonate groups will be obtained.

T2: Characterization of solid supports: characteristic parameters such as size and size distribution of pores as well as permeability and surface area will be determined using complementary methods. Correlations between synthesis conditions (time and intensity of irradiation, monomer ratio …) and morphology of the porous monoliths will be established.

T3: Application of porous monoliths-containing microsystem to the analysis of phosphopeptides: the objective will be on the one hand, to successfully achieve the separation of peptides as function of their site and degree of phosphorylation and on the other hand, to quantify the efficiency of the monolith-assisted method in terms of enrichment factor. 

[1] Hanger, P.D., Anderton, B.H., Nobe, W. Trends Mol. Med. Vol 15 (3), 112-119 (2009)

[2] Thingholm, T.E., Jensen, O.N., Larsen. M. Meth. Prot. Vol 527, 67-78 (2009)

[3] Namera, A., Nakamoto, A., Saito, T., Miyazaki, S.J. Sep. Sci. Vol 34, 901-924 (2011)

[4] Dziomba S., Araya-Farias M., Taverna M., Guerrouache M., Carbonnier B., Tran N.T. Analytical and Bioanalytical chemistry, 2017, 409, 2155-2162

[5] Araya-Farias M., Taverna T., Carbonnier N., Woytasik M., Bayle F., Guerrouache M., Cao Hong Ha, Tran N.T. Polymer, 2015, 66, 249-258.

[6] Araya-Farias M., Dziomba S., Carbonnier B., Guerrouache M., Ayed I., Aboud N., Taverna M., Tran N.T. Analyst, 2017, 142, 485-494.


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