Mass spectrometric detection of KRAS protein mutations using molecular imprinting

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.

Fig. 1. Method development of KRAS-capturing nanoMIPs; LC-MS was used to analyse the: (A) elution of KRAS bridged peptide from nanoMIPs using 10% acetonitrile/10% formic acid (elution is preceded by 5 washes with PBS); (B) elution of KRAS peptide from the nanoMIPs in plasma and water (elution is preceded by 5 washes with PBS), and (C) reusability of MIPs as demonstrated by repeated capture of KRAS.

Fig. 2. LC-SRM chromatograms for (A) KRAS WT peptide (SRM transitions 478.30 → 743.44, 644.37, 545.30) and (B) stable isotopically labelled standard for KRAS WT peptide (SRM transitions 482.31 → 751.46, 652.39, 553.32) from H1650 cell lysate using on bead digests; and (C) mutant peptide (SRM transitions 493.31 → 773.45, 674.38, 575.31) and (D) stable isotopically labelled standard for G12S (SRM transitions 497.31 → 781.47, 682.397, 583.33) from A549 cell lysate on-bead digests. Multiple transitions sharing the same chromatographic profile are shown for each peptide. Vertical dotted lines indicate an automatically selected peak by the software on predicted parameters.

Fig. 3. Quantification of KRAS WT 6–16 peptide in pancreatic cancer (blue) and NSCLC (red) plasma samples. The figure shows a spread of KRAS measurements for each cancer.

Fig. 4. LC-SRM detection of mutated KRAS proteins in pancreatic cancer patients. Each Panel A to D are derived from different cancer patients where a mutation is tentatively identified. Within each panel, the transitions for the analyte are shown on the upper chromatogram whilst the relevant transitions for stable isotope labelled (SIL) standards are shown in the lower chromatogram. Panel A to D show tentative assignments for G → C, G → D, G → S and G → A respectively.

Fig. 5. Western blot analysis for detection of KRAS using a KRAS primary antibody. (Lane 1) molecular weight marker, (lanes 2–5,7) NSCLC patient plasma, (lane 6) Pancreatic cancer plasma, (lane 8) a healthy control patient plasma, (lane 9) HCT-N cells and (lane 10) HT-29 cells.

Fig. 6. Identification of peptide sequences exposed on protein surfaces using molecular imprinting and mass spectrometry.

Fig. 7. Epitope discovery of nanoMIPs with recombinant KRAS protein (A) epitope sites as identified by nanoMIP directed MS proteomics, (B) crystal structure of KRAS4B (C) surface representation of chemical shift changes on KRAS4B in the same orientation as B. (D) 180° rotation of image C.