On the instrumentation front, two streams of technology have emerged as critical to the biomarker effort. On the one hand, increasing resolution, sensitivity, and speed of high-end mass spectrometers now enables the detection of tens of thousands of tryptic peptides (and by inference thousands of proteins from which they came) in complex biological samples.
On the other hand, a separate stream of quantitative MS technology, measuring preselected peptide ions based on two mass parameters (parent peptide and a specific sequence fragment, in so called multiple-reaction monitoring, or MRM, mode) provides a capability to accurately (~10% CV) quantitate 100 or more peptides at much higher throughput.
The sensitivity of multiplex MRM technology can be extended down to the ng/mL level and below by abundant protein depletion combined with limited fractionation or specific capture of the target peptides on antipeptide antibodies (the SISCAPA technique), covering a majority of the known biomarker proteins detected in blood plasma.
It is now clear that a functional biomarker pipeline needs both of these approaches: shotgun methods to search large numbers of peptides and proteins for potential disease-related differences, albeit with a high false-discovery rate, and MRM methods to construct accurate high-throughput assays to be applied to relevant Zolg-scale sample sets to verify performance in real populations.
Evaluating, optimizing, and implementing these and other recent advances are critical to solving the general biomarker problem. To do so, however, requires enlarging the focus of our efforts from technology-centric academic proteomics to a multidisciplinary (though possibly virtual) biomarker pipeline.
Productive relationships must be forged between disparate technology platforms and between technological, medical/biological, and statistical specialties. The U.S. National Cancer Institute is attempting to create a nucleus for this new approach in its Clinical Proteomic Technology Assessment for Cancer program within the broader Clinical Proteomic Technologies for Cancer initiative.
Beginning with a critical evaluation of existing technology and methods, the CPTAC teams have designed and carried out true multisite reproducibility studies of both approaches: shotgun unbiased discovery and targeted MRM assays. The results, recently presented and now submitted for publication, are revealing.
As has been expected based on earlier, less well-controlled studies (e.g., the HUPO plasma proteome exercise), the shotgun approaches produce a statistical samples of of the peptides in the proteome under study and thus often show significant differences in the sets of peptides from run to run both within and between laboratories (with greater similarity at the protein level).
The need for replicate runs to approach asymptotic completeness in proteome coverage is thus an inherent statistical feature of the method. The targeted MRM assays, on the other hand, derived from a widely used accurate quantitation approach for small molecules, can yield results that are accurate, reproducible (in this case across eight sites), and of wide dynamic range provided we restrict attention to a set of up to several hundred prespecified peptides.
These studies have confirmed the roles and fitness of these two approaches for discovery and verification of candidates in the biomarker pipeline, and provide confidence that both can be practiced effectively in multiple laboratories.
In parallel, it appears that MS-based measurements can deliver high-quality results in clinical laboratories as well. A specific example highlighting these issues is the clinical assay for plasma thyroglobulin, a thyroid-specific protein used to detect recurrence of thyroid cancer in patients whose diseased thyroids have been removed.
Andrew Hoofnagle, M.D., Ph.D., recently demonstrated an MS-based SISCAPA assay for peptides from thyroglobulin designed to circumvent several well-known and high-prevalence interferences plaguing the existing commercial immunoassays for this protein.
The prospects for major progress in protein biomarkers in readily accessible bodily fluids thus appear considerably brighter than even a year or two ago. The clinical and economic value of early detection of diseases like cancer, COPD, or Alzheimer’s is so great that once a real biomarker pipeline is proven, and the odds shifted from no chance to merely long, a strong case exists for more equal emphasis on the development of biomarkers and drugs.