The measurement of KRAS G12 mutants using multiplexed selected reaction monitoring and ion mobility mass spectrometry

Abstract

Rationale There is a considerable clinical demand to determine key mutations in genes involved with cancer which necessitates the deployment of highly specific and robust analytical methods. Multiplex liquid chromatography with selected reaction monitoring (LC/SRM) assays offer the ability to achieve quantitation down to levels expected to be present in clinical samples. Ion mobility mass spectrometry (IMS/MS) assays can provide increased peak capacity and hence separation in an extremely short time frame, and in addition provide physicochemical data regarding the collision cross‐section of an analyte which can be used in conjunction with the m/z value of an ion to increase detection specificity. Methods For LC/SRM, unlabelled peptides and corresponding stable‐isotope‐labelled standards were spiked into digested human plasma and analysed using ultrahigh‐performance liquid chromatography (UHPLC) coupled to a triple quadrupole mass spectrometer to enable the generation of analyte‐specific calibration lines. Synthetic unlabelled peptides were infused into a Synapt G2 mass spectrometer for travelling wave ion mobility separation and TWCCSN2 values were derived from comparison with previously generated TWCCSN2 calibration values. Results Linear calibration lines (0.125 to 25 fmol/μL) were established for each of the KRAS peptides. UHPLC separated the peptides and hence enabled them to be split into different retention time functions/windows. This separation enabled detection of three or four transitions for each light and heavy peptide with at least 10 points per peak for accurate quantitation. All six KRAS G12 peptides were separated using IMS/MS, enabling precise TWCCSN2 values to be determined. Although some of the G12 peptides chromatographically co‐eluted, all the peptides were distinguished by m/z, retention time and/or drift time. Conclusions This study advocates that LC/SRM and IMS/MS could both be used to identify single amino acid substitutions in KRAS as an alternative to commonly used methods such as circulating tumour DNA analysis.

The addition of FAIMS increases targeted proteomics sensitivity from FFPE tumor biopsies

“…The addition of an ion mobility step to an SRM method has been described previously 19 – 21 . One report describes a method combining IMS with quadrupole time-of-flight fragment ion detection for the quantitation of host cell proteins in protein biopharmaceutical products 22 ; in this case the IMS was integral to the mass spectrometer, prohibiting a comparison of the same method with and without ion mobility.…”

Mass spectrometric detection of KRAS protein mutations using molecular imprinting

“… 11 Calibration lines were established for 6 of the main mutations and other RAS forms. 34 For every sample, we were able to detect and measure WT KRAS ( Fig. 3 ), and to the best of our knowledge, this is the first occasion that the gene product of KRAS has been measured in cancer patient plasma using LC-SRM.…”

The addition of FAIMS Increases Targeted Proteomics Sensitivity from FFPE Tumor Biopsies

“…The addition of an ion mobility step to an SRM method has been described previously [19][20][21] . One report describes a method combining IMS with quadrupole time-of-flight fragment ion detection for the quantitation of host cell proteins in protein biopharmaceutical products 22 ; in this case the IMS was integral to the mass spectrometer, prohibiting a comparison of the same method with and without ion mobility.…”

“…Although sample fractionation or enrichment strategies can be applied to reduce complexity and non-specific background signals 17,18 , they also decrease the overall throughput and, more importantly, are not feasible when there is limited availability of clinical samples. Ion mobility spectrometry (IMS) can reduce complexity in the gas phase without having to fractionate samples in advance and has been applied to peptide quantitation by SRM [19][20][21][22] .…”