The Bottleneck in the Cancer Biomarker Pipeline and Protein Quantification through Mass Spectrometry–Based Approaches: Current Strategies for Candidate Verification

Abstract

Although robust discovery-phase platforms have resulted in the generation of large numbers of candidate cancer biomarkers, a comparable system for subsequent quantitative assessment and verification of all candidates is lacking. Established immunoassays and available antibodies permit analysis of small subsets of candidates; however, the lack of commercially available reagents, coupled with high costs and lengthy production and purification times, have rendered the large majority of candidates untestable. Mass spectrometry (MS), and in particular multiple reaction monitoring (MRM)-MS, has emerged as an alternative technology to immunoassays for quantification of target proteins. Novel biomarkers are expected to be present in serum in the low (microg/L-ng/L) range, but analysis of complex serum or plasma digests by MS has yielded milligram per liter limits of detection at best. The coupling of prior sample purification strategies such as enrichment of target analytes, depletion of high-abundance proteins, and prefractionation, has enabled reliable penetration into the low microgram per liter range. This review highlights prospects for candidate verification through MS-based methods. We first outline the biomarker discovery pipeline and its existing bottleneck; we then discuss various MRM-based strategies for targeted protein quantification, the applicability of such methods for candidate verification, and points of concern. Although it is unlikely that MS-based protein quantification will replace immunoassays in the near future, with the expected improvements in limits of detection and specificity in instrumentation, MRM-based approaches show great promise for alleviating the existing bottleneck to discovery.