Targeted Mass Spectrometry Analyses of Somatic Mutations in Colorectal Cancer Specimens Using Differential Ion...

Area of Science:

• Biochemistry
• Oncology
• Analytical Chemistry

Background:

• K-Ras and B-Raf mutations are key biomarkers in colorectal cancer (CRC) for predicting response to anti-EGFR therapies.
• Accurate identification of these mutations is vital for personalized treatment strategies and improved patient outcomes.

Purpose of the Study:

• To enhance the sensitivity and specificity of detecting low-abundance K-Ras and B-Raf mutations in CRC samples.
• To demonstrate the utility of combining parallel reaction monitoring (PRM) with high-field asymmetric waveform ion mobility (FAIMS) for mass spectrometry-based mutation analysis.

Main Methods:

• Integration of high-field asymmetric waveform ion mobility (FAIMS) with targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) and parallel reaction monitoring (PRM).
• Application of the developed method for the detection and quantitation of specific mutations (B-Raf V600E, K-Ras G12D, G13D, G12V) without immunoaffinity enrichment.

Main Results:

• FAIMS significantly reduced interfering ions and improved precursor ion purity in targeted LC-MS/MS analyses.
• A 3-fold improvement in the detection limit for K-Ras and B-Raf mutated peptides was achieved.
• FAIMS enabled unambiguous identification of isomeric K-Ras G12D and G13D peptides.

Conclusions:

• The combined PRM and FAIMS approach offers enhanced sensitivity for detecting low-abundance mutations in CRC.
• This method provides a robust platform for the precise identification and quantitation of actionable mutations in clinical specimens.
• FAIMS-enhanced mass spectrometry represents a valuable tool for advancing precision oncology in colorectal cancer.