The measurement of KRAS G12 mutants using multiplexed selected reaction monitoring and ion mobility mass spectrometry.
Rapid Commun Mass Spectrom
; 34 Suppl 4: e8657, 2020 Sep.
Article
en En
| MEDLINE
| ID: mdl-31800120
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 TW CCSN2 values were derived from comparison with previously generated TW CCSN2 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 TW CCSN2 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.
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Espectrometría de Masas
/
Proteínas Proto-Oncogénicas p21(ras)
/
Mutación
Tipo de estudio:
Guideline
Límite:
Humans
Idioma:
En
Revista:
Rapid Commun Mass Spectrom
Año:
2020
Tipo del documento:
Article
País de afiliación:
Reino Unido