Mutant proteins as cancer-specific biomarkers

Abstract

Cancer biomarkers are currently the subject of intense research because of their potential utility for diagnosis, prognosis, and targeted therapy. In theory, the gene products resulting from somatic mutations are the ultimate protein biomarkers, being not simply associated with tumors but actually responsible for tumorigenesis. We show here that the altered protein products resulting from somatic mutations can be identified directly and quantified by mass spectrometry. The peptides expressed from normal and mutant alleles were detected by selected reaction monitoring (SRM) of their product ions using a triple-quadrupole mass spectrometer. As a prototypical example of this approach, we demonstrated that it is possible to quantify the number and fraction of mutant Ras protein present in cancer cell lines. There were an average of 1.3 million molecules of Ras protein per cell, and the ratio of mutant to normal Ras proteins ranged from 0.49 to 5.6. Similarly, we found that mutant Ras proteins could be detected and quantified in clinical specimens such as colorectal and pancreatic tumor tissues as well as in premalignant pancreatic cyst fluids. In addition to answering basic questions about the relative levels of genetically abnormal proteins in tumors, this approach could prove useful for diagnostic applications.

Fig. 1.

Schematic of the approach.

Fig. 2.

IP of Ras proteins. An antibody directed against a common epitope of all three forms of mutant and WT forms of Ras (K-Ras, N-Ras, and H-Ras) was used to immunoprecipitate the indicated amounts of protein in SW480 cell lysates. Western blots were performed using an HRP-conjugated monoclonal antibody to K-Ras. Ten nanograms of recombinant K-Ras protein were loaded on the right-most lane of each gel for comparison purposes. The “input lysate” and “lysate after IP” lanes contained 4% of the proteins used for IP; all the eluted protein and protein remaining on beads were loaded into the corresponding lanes.

Fig. 3.

Extracted ion chromatograms of ¹³C/¹⁵N-labeled synthetic peptides. The retention times of the indicated peptides are shown above the peaks in A–C, and the insets at the right of each panel represent an expanded view. Asterisks indicate the heavy isotope (¹³C₆/¹⁵N₂)-labeled lysine. D–F illustrate the relationship between the amount of peptide injected into the mass spectrometer and the integrated intensity of the transitions. The b and y peaks indicate the detected intensities of b ions and y ions (as designated in Table S1).

Fig. 4.

SRM of endogenous proteins from SW480 cells. (A and B) Extracted ion chromatograms of transitions from (A) exogenously added heavy isotope-labeled WT peptide and (B) corresponding endogenous WT peptide, illustrating the identical retention times. (C and D) Extracted ion chromatograms of the (C) exogenous and (D) endogenous mutant peptides. In each panel, the inset at the right represents an expanded view of the major peaks. Asterisks indicate the heavy isotope (¹³C₆/¹⁵N₂)-labeled lysine.

Fig. 5.

SRM of endogenous proteins from a colorectal tumor obtained at surgery. (A) Integrated intensities of the exogenously added mutant peptide and the endogenous mutant peptide from the tumor, as indicated. The integrated intensities correspond to the sum of the peak areas of the transitions described in Table S1, which are shown in B for the endogenous peptide. The asterisk indicates the heavy isotope (¹³C₆/¹⁵N₂) labeled lysine.