Molecularly imprinted polymers synthesized via template immobilization on fumed silica nanoparticles for the enrichment of phosphopeptides. Issue 3 (12th October 2017)

• Record Type: Journal Article
• Authors: Duarte, Mariana; Subedi, Prabal; Yilmaz, Ecevit; Marcus, Katrin; Laurell, Thomas; Ekström, Simon
• Other Names: Spivak David A. guestEditor.
• Is Part Of: Journal of molecular recognition. Volume 31:Issue 3(2018)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2017-10-12
• Subjects: fumed silica nanoparticles -- molecularly imprinted polymers -- phosphopeptide enrichment -- pore‐size distribution -- surface imprinting; Molecular recognition -- Periodicals; Models, Molecular -- Periodicals; Molecular Conformation -- Periodicals; Molecular Sequence Data -- Periodicals; Molecular Structure -- Periodicals; Carrier Proteins -- Periodicals; 572.8
• DOI: 10.1002/jmr.2677
• Languages: English
• ISSNs: 0952-3499

Abstract

Abstract: Phosphorylation is a protein post‐translational modification (PTM) that plays an important role in cell signaling, cell differentiation, and metabolism. The hyper phosphorylated forms of certain proteins have been appointed as biomarkers for neurodegenerative diseases, and phosphorylation‐related mutations are important for detecting cancer pathways. Due to the low abundance of phosphorylated proteins in biological fluids, sample enrichment is beneficial prior to detection. Thus, a need to find new strategies for enriching phosphopeptides has emerged. Molecularly imprinted polymers (MIPs) are synthetic polymeric materials manufactured to exhibit affinity for a target molecule. In this study, MIPs have been synthesized using a new approach based on the use of fumed silica as sacrificial support acting as solid porogen with the template (phosphotyrosine) immobilized on its surface. Phosphotyrosine MIPs were tested against a mixture of peptides and phosphopeptides by performing micro‐solid phase extraction using MIPs (μMISPE) packed in a pipette tip. First, the capability of the materials to preferentially enrich phosphopeptides was evaluated. In a next step, the enrichment of phosphopeptides from a whole‐cell lysate of human embryonic kidney (HEK) 293T cells was performed. The eluates were analyzed using MALDI‐MS in the first case and with nano‐HPLC‐ESI‐MS/MS in the second case. The results showed that the MIPs provided affinity for phosphopeptides, binding preferentially to multi‐site phosphorylated peptides. The MIPs could enrich phosphopeptides in over 10‐fold compared with the number of phosphopeptides found in a cell lysate without enrichment. Abstract : Phosphotyrosine‐imprinted polymers with a narrow and reproducible distribution of pore sizes were synthesized in the absence of solvent using fumed silica. The template phosphotyrosine was first immobilized on the surface of aminopropyl‐silica nanoparticles. The resulting polymers were tested as sorbents for the enrichment of phosphopeptides. The binding and elution of phosphopeptides was evaluated using a mixture of known composition and a crude biological sample. Results have shown a selectivity trend towards multiside phosphorylated peptides by the molecularly imprinted polymers.