Would you prefer multiple reaction monitoring or antibodies with your biomarker validation?

The past several years have seen the proteomics community working fervently to discover proteins that can be used as biomarkers of diseases such as cancer, neuro logical disorders and others. While the scientific community can debate the relative success of this continuing endeavor, the lack of clinically useful protein biomarkers that have been reported from these efforts has not prevented investigators from looking forward toward developing methods that are necessary to verify the clinical utility of a biomarker. As wonderfully described by Steve Carr, the four phases of producing a novel protein biomarker are discovery, qualification, verification and validation [1]. One of the most striking features as the process moves from discovery to validation is the inverse relationship between the number of analytes and samples that are measured. While the discovery phase may require only tens of samples to be scanned for thousands of analytes, the validation phase will require only a few analytes to be measured in thousands of samples. During the validation phase, clinical assay development may proceed concurrently. Mass spectrometry (MS), combined with a variety of sample preparation and separation techniques, has been the driving force in the biomarker discovery phase [2–4]. The question is how big a role MS will play in the qualification, verification and validation phases. Conventional thinking suggests that antibodies (or other types of affinity reagents) provide the most expedient means of validating the usefulness of a potential biomarker. Antibody-based tests such as immunohistochemistry (IHC) and ELISA are routinely performed, and a wide range of scientists feel comfortable in the results obtained using these methods. There is, however, another resurgent method that is being touted as an alternative to antibody-based tests, multiple reaction monitoring (MRM)-MS. This method uses triple quadrupole MS to specifically target and quantify proteins of interest in a complex mixture. Ions corresponding to the mass of the biomolecule of interest are isolated within the mass spectrometer. The molecule is then fragmented and a selection of these products is monitored and used for identification and quantitation purposes. The MRM-MS approach provides exquisite specificity and does not suffer from issues related to cross-reactivity with other molecules. If designed properly, only the targeted molecule will be measured by the mass spectrometer. In addition, the method does not require a highly specific antibody (or antibodies) and permits absolute quantitation through the addition of known quantities of isotopically labeled molecules to the mixture. The increasing popularity of MRM-MS was evident at the past 2008 meeting of the American Society of Mass Spectrometry (ASMS) by the number of oral and poster presentations that highlighted this method. The question that one has to answer is: will MRM-MS replace antibody-based testing in the validation of biomarkers? Based on many of the presentations at the past ASMS meeting, one would think that that is the case. However, a number of presentations simply highlighted the advantages of MRM-MS by focusing on the disadvantages Haleem J Issaq, PhD Principal Scientist and Deputy Director, Laboratory of Proteomics and Analytical Technologies, Advanced Technology Program, SAIC-Frederick Inc., National Cancer Institute at Frederick, Frederick, MD 21702-1201, USA Tel. : +1 301 846 7186 Fax: +1 301 846 6037 issaqh@mail.ncifcrf. gov