Molecularly imprinted polymers synthesized via template immobilization on fumed silica nanoparticles for the enrichment of phosphopeptides

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 phosphorylationrelated 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. MS-based studies of protein phosphorylation are dependent on sample preparation steps that enrich the phosphorylated peptides prior to analysis.

Phosphorylation Time‐Course Study of the Response during Adenovirus Type 2 Infection

“…Using MS‐based approaches, thousands of phosphorylations can be monitored in a sample, [ 22,23 ] but enrichment steps are needed during sample preparation to separate off high abundant native peptides that mask the detection of modified peptides. [ 24–26 ] A variety of different enrichment strategies are used, [ 24,27,28 ] and among them, metal oxides such as titanium dioxide (TiO 2 ) and zirconium dioxide are preferred due to their great tolerance to buffers and detergents. The combination of these enrichment procedures with sensitive and high‐resolution MS enables the identification and characterization of a high number of proteins and modifications simultaneously.…”

Review on molecularly imprinted polymers with a focus on their application to the analysis of protein biomarkers

“…Secondly, this technique is useful for insoluble templates, since they do not need to be incorporated in the solvent. Finally, the immobilisation of template via chemical bonds enables complete and easy template removal after polymerisation without the need for excessive washing cycles [107,108].…”

Molecularly Imprinted Materials for Selective Biological Recognition

“…Molecular recognition strategies based on living cells mainly include whole cell imprinting. The molecular imprinted polymer receptors are capable of specific interacting or recognizing of living cells, making them attractive for bio‐related applications such as cell capture, cell imaging, cell differentiation, and inhibition of cancer cells . For example, the whole cell‐imprinted substrates based on chondrocytes, tenocytes, and semi‐fibroblasts as templates were prepared by Bonakdar et al to demonstrate their differentiation, redifferentiation, and transdifferentiation …”