An improved quantitative mass spectrometry analysis of tumor specific mutant proteins at high sensitivity

Abstract

New disease specific biomarkers, especially for cancer, are urgently needed to improve individual diagnosis, prognosis, and treatment selection, that is, for personalized medicine. Genetic mutations that affect protein function drive cancer. Therefore, the detection of such mutations represents a source of cancer specific biomarkers. Here we confirm the implementation of the mutant protein specific immuno-SRM (where SRM is selective reaction monitoring) mass spectrometry method of RAS proteins reported by Wang et al. [Proc. Natl. Acad. Sci. USA 2011, 108, 2444-2449], which exploits an antibody to simultaneously capture the different forms of the target protein and the resolving power and sensitivity of LC-MS/MS and improve the technique by using a more sensitive mass spectrometer. The mutant form G12D was quantified by SRM on a QTRAP 5500 mass spectrometer and the MIDAS workflow was used to confirm the sequence of the targeted peptides. This assay has been applied to quantify wild type and mutant RAS proteins in patient tumors, xenografted human tissue, and benign human epidermal tumors at high sensitivity. The limit of detection for the target proteins was as low as 12 amol (0.25 pg). It requires low starting amounts of tissue (ca.15 mg) that could be obtained from a needle aspiration biopsy. The described strategy could find application in the clinical arena and be applied to the study of expression of protein variants in disease.

Large-Scale Mass Spectrometric Detection of Variant Peptides Resulting from Nonsynonymous Nucleotide Differences

“…Related to this finding, we also investigated the feasibility of quantifying differential allelic expression on a large scale. Previously, SRM methods employing stable isotope labeled peptide standards were developed to quantify three allelic peptide pairs 52 and a small number of related mutant peptides 48, 53 . Here, we presented preliminary label-free quantification of allele-specific expression based on the integrated MS 1 extracted ion chromatograms from 51 allelic peptide pairs.…”

Advances in targeted proteomics and applications to biomedical research

“…Antibody‐based immunoaffinity enrichment coupled to SRM (i.e., immuno‐SRM) has emerged as a promising technology for sensitive precise quantification of target proteins in complex matrices . For example, it was applied for quantification of mutant RAS (G12D) at LOD of 12 amol (i.e., 0.25 pg) or 240 amol/mg of total protein in patient tumors by anti‐RAS protein antibody at the protein‐level enrichment and FGF15 at LOD of 0.1 ng/mL in mouse plasma by anti‐peptide antibody at the peptide‐level enrichment (i.e., SISCAPA, stable isotope standard and capture by the anti‐peptide antibody) . Recently, the development of immuno‐SRM has been primarily focused on multiplexing , assay sensitivity , and rapid cost‐effective generation of antibodies .…”

The clinical impact of recent advances in LC-MS for cancer biomarker discovery and verification

“…The wild-type and mutant KRAS proteins in patient tumor and xenograft human tissue were quantified, and the LOD was as low as 0.24 fmol/mg of total protein. [101] The Reubsaet group reported their work using immunocapture-SRM. [102,116] Recently, they developed a multiplexing immunocapture technique, in which two kinds of antibody beads were utilized to co-extract different targeted markers simultaneously.…”