Abstract
RationaleThere 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.MethodsFor 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.ResultsLinear 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.ConclusionsThis 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.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
