Abstract
Cancer is a disease of cellular evolution where single base changes in the genetic code can have significant impact on the translation of proteins and their activity. Thus, in cancer research there is significant interest in methods that can determine mutations and identify the significant binding sites (epitopes) of antibodies to proteins in order to develop novel therapies. Nano molecularly imprinted polymers (nanoMIPs) provide an alternative to antibodies as reagents capable of specifically capturing target molecules depending on their structure. In this study, we used nanoMIPs to capture KRAS, a critical oncogene, to identify mutations which when present are indicative of oncological progress. Herein, coupling nanoMIPs (capture) and liquid chromatography-mass spectrometry (detection), LC-MS has allowed us to investigate mutational assignment and epitope discovery. Specifically, we have shown epitope discovery by generating nanoMIPs to a recombinant KRAS protein and identifying three regions of the protein which have been previously assigned as epitopes using much more time-consuming protocols. The mutation status of the released tryptic peptide was identified by LC-MS following capture of the conserved region of KRAS using nanoMIPS, which were tryptically digested, thus releasing the sequence of a non-conserved (mutated) region. This approach was tested in cell lines where we showed the effective genotyping of a KRAS cell line and in the plasma of cancer patients, thus demonstrating its ability to diagnose precisely the mutational status of a patient. This work provides a clear line-of-sight for the use of nanoMIPs to its translation from research into diagnostic and clinical utility.
Highlights
Precision medicine in cancer therapy requires highly specific, reproducible and quantitative methods for the identification and quantitation of molecules that indicate the mutational status of a patient, help informing the clinician about the most appropriate therapy.[1,2,3] Ideally, these methods should be minimally invasive, reproducible, and cost effective and avoid potentially complicated procedures such as tissue biopsy.[4]
We describe the synthesis and application of Molecularly imprinted polymers (MIPs) as a diagnostic tool to recognise the C-terminus of the KRAS protein, which is conserved in both the wild type (WT) and mutated protein
NanoMIPs were tested on their ability to capture a synthetic model KRAS molecule which contained 35 amino acid sequence including 22 amino acids from the N-terminus of KRAS and 13 amino acid sequence of C-terminus (LVVVGAGGVGKSALTIQLIQNHTPGCVKIKKCIIM)
Summary
Precision medicine in cancer therapy requires highly specific, reproducible and quantitative methods for the identification and quantitation of molecules that indicate the mutational status of a patient, help informing the clinician about the most appropriate therapy.[1,2,3] Ideally, these methods should be minimally invasive, reproducible, and cost effective and avoid potentially complicated procedures such as tissue biopsy.[4] Current methods to determine the mutation status of a patient rely on polymerase chain reaction (PCR) of tissue samples, which can take up to a week to obtain results.[5] there is a clear clinical need to establish a method which is non-invasive, highly specific and will provide results in a reasonably short turnaround time. Ras proteins act as molecular switches by alternating between an active GTP-bound state and an inactive GDP-bound conformation. Ras proteins constitute major players in the MAPK pathway and are activated in response to ligand binding to Epidermal Growth Factor Receptor (EGFR).[8] Ligand binding to EGFR leads to dimerisation of the receptor, changing its conformation so that growth factor receptor-bound protein 2 (GRB2) can bind. GRB2 recruits son of sevenless (SOS), a guanine nucleotide exchange factor (GEF) which dissociates guanine nucleotides from KRAS, allowing the more abundant GTP to bind and activate KRAS.[9,10] Active KRAS binds to and enables activation of Communication
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