The project goal is that of preparing optimized materials for enrichment of peptides with phosphorylation, glycosylation, and/or sulfation modifications. These peptides have important biological activities but the direct analysis of protein post translational modifications (PTMs) without enrichment is impaired by suppression of non-modified peptides. The materials to be developed are magnetic nanoparticle functionalized with polymers based on 2-acrylamido-2-methylpropane sulfonic acid (AMPS) monomer with cross linker ethylene glycol dimethylacrylate (EGDMA) in presence of dimethyl sulfoxide as solvent and acetonitrile and/or 1,4-butanediol as porogens. Polymer preparation will be optimizated by Box-Behnken design (BBD) for the monomer-to-crosslinker ratio and the porogen composition. By this approach, the hydrophilicity of the surface and pore structure is expected to be modulated, to determine the most suited ones for enrichment of peptides with the target PTMs. The materials, prepared according to this experimental design, will be used for enrichment evaluation. Standard glycoprotein fetuin, phosphoprotein casein and sulfopeptides will initially be used for enrichment, then the most promising materials will be applied to serum protein digests. A combined simultaneous enrichment will also be tested if good results can be obtained from enrichment experiments on single PTM.
The project has the goal of producing materials, based on 2-acrylamido-2-methylpropane sulfonic acid (AMPS) monomer, for enrichment of peptides with PTMs. This monomer was never used for preparation of materials for this purpose but can be potentially very successful due to hydrophilicity of products, which is expected to positively affect the enrichment efficiency.
The structure of the material will be optimized by design of experiment approach, for optimization of the preparation and composition. Although many materials are developed for enrichment of PTMs in proteomics, the use of a design of experiment optimization is uncommon [1]. Such an approach can improve the performance of the enrichment process by fine tuning the material features, in this case surface hydrophilicity and pore structure. By this approach, after choosing a polymer composition, the most suitable structure for the specific purpose can be determined. This is important as the fine structure of materials can affect the enrichment performance, as previously described, for instance, for carbon nanostructures [2].
The materials will be optimized for glycopeptide, phosphopeptide or sulfopeptide enrichment. As a simple goal, a new material for the individual enrichment of these PTMs will be provided. If there are already plenty of materials for enrichment of phosphopeptides and glycopeptides, the same is not for sulfopeptides. Protein sulfation, as described in the state of the art section, is particularly neglected in the proteomic community, due to difficulty in detection of sulfopeptides by LC-MS proteomics. However, our research group has been working on the issue, and has developed an enrichment method for sulfopeptide standard compounds based on Fe3+-IMAC. Moreover, standard peptides with the same sequence but different modification (tyrosine sulfation or phosphorylation) were used to devise a MS strategy suitable to study the sequence of these peptides and differentiate them by MS and MS/MS analysis. The data are unpublished, yet, and were obtained within the framework of EPIC-XS project (project number 0000104) at the Utrecht University in combination with our research group. These data show such information can be achieved by combining positive and negative ion ionization MS/MS analysis. As such, the extension to sulfopeptides, as devised in the presented project, is completely new for the field and could potentially provide an improved strategy for characterization of these peptides in complex matrices. As described in the literature section, methods for sulfopeptides are really scarce, which make this contribution particularly interesting, especially if unknown sulfopeptides will be identified from the serum samples.
Finally, the type of polymer used for development is expected to be potentially able to enrich all these modifications together, which is a great improvement over previously described methods. In fact, if methods for the simultaneous enrichment of phosphopeptides and glycopeptides (mostly sialopeptides) were described in the literature [3], the simultaneous enrichment of sulfopeptides would be completely new.
[1] L. Pieroni, F. Iavarone, A. Olianas, V. Greco, C. Desiderio, C. Martelli, B. Manconi, M. T. Sanna, I. Messana, M. Castagnola, T. Cabras, Journal of Separation Science 2020, 43:313
[2] S. Piovesana, D. Iglesias, M. Melle-Franco, S. Kralj, C. Cavaliere, M. Melchionna, A. Laganà, A. L. Capriotti, S. Marchesan, Nano Research 2020, 13:380
[3] K.-C. Cho, L. Chen, Y. Hu, M. Schnaubelt, H. Zhang, ACS Chemical biology 2019, 14:58