Immuno Tandem Mass Spectrometry (iMASS~(2)) Assay for Clinical Proteomics: Development and Application of iMASS~(2) for EGFR Diagnosis

摘要

The epidermal growth factor receptor (EGFR) is highly expressed in a variety of cancer tumors, and is therefore an important biomarker and target for cancer therapy. We have developed a novel peptide-based immuno tandem mass spectrometry (iMASS~(2)) diagnostic assay for highly sensitive, specific, and quantitative analysis of EGFR. This peptide-based assay offers several advantages including the elimination of extensive protein separation steps and the ability to capture low abundance peptides in complex samples, thus providing a wide dynamic range of detection. Anti-peptide antibodies are immobilized on inexpensive affinity beads, which eliminates the need for special surface chemistry and allows for less stringent storage conditions after antibody immobilization for reduced denaturation of the antibody in solution. Customized anti-peptide antibody was immobilized to CNBr-activated sepharose beads according to the manufacturer's protocol. Sample containing the target protein alone, or highly expressed cell lysate was digested with trypsin; the digest was then incubated with the immobilized anti-peptide antibody beads. Stable-isotope labeled derivatives of the epitope-containing peptide, synthesized at the UNC Peptide Synthesis Facility (UNC-PSF), were added as internal standards for absolute quantitation. Finally, the beads were placed on the MALDI target with matrix solution for MS and MS/MS analysis.