BACKGROUND: 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

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... candidates untestable. CONTENT: 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 (g/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 highabundance 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. SUMMARY: 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, MRMbased approaches show great promise for alleviating the existing bottleneck to discovery. Despite the recent progress toward understanding of cancer etiology and the implementation of preventative measures and novel therapeutic modalities, cancer remains a major disease burden. According to the International Agency for Research on Cancer, in 2008 there were approximately 12.4 million new cancer cases and 7.6 million cancer-related deaths worldwide (1 ). Within the US, this translates to approximately 1.5 million new cases and half a million deaths expected for 2009 (2 ). Although recent trends show a slight decline in incidence and mortality rates for cancers affecting some of the more prevalent cancer sites, such as lung, prostate, colon, and breast, the overall burden of cancer will likely increase in the near future as an increasing percentage of the world's population reaches old age (2 ). To alleviate both the economic and social costs posed by cancer, there is an urgent need to discover and validate novel cancer biomarkers suitable for early disease diagnosis and optimal patient management. Biomarkers are generally quantifiable molecules or processes indicative of a certain biological state or condition, and in the context of cancer, various molecular analytes (such as DNA, mRNA, microRNA, and proteins) and physiological processes (e.g., angiogenesis and proliferation) have proven useful for cancer detection and management (3-6 ). All of these genetic and molecular alterations, however, tend to ultimately culminate in the aberrant (increased or decreased) expression of protein products. As a result, proteins and the study of cancer proteomics are a potential gold mine for discovery of novel biomarkers. In this review we outline a widely used pipeline for discovery of novel cancer biomarkers, the existing "bottleneck" in moving markers from discovery phases to the clinical arena,