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The enormous potential of biomarkers to revolutionize clinical practice and improve patient care has been well documented (1,2). Molecular-based diagnostic and prognostic tests, particularly those aimed at protein analytes, could be used to detect disease earlier enabling treatment to start sooner and possibly cure rather than delay further injury or death. They could also be used to more accurately stage disease and to predict response to therapy, thereby helping select the correct treatment. Biomarkers can also be used to stratify patients for assessment of new drug therapies and to serve as surrogate endpoints in early phase drug trials, thereby lowering the overall cost of drug development and resulting in more effective treatments. Given their high potential therapeutic and financial impact it is, on the surface, surprising that so few new protein biomarkers have been introduced into widespread clinical use recently. In fact, only five new protein markers have been FDA approved for measurement in plasma or serum in the last 5 years.
The reasons for the dearth of new protein biomarkers are gradually becoming clearer - they relate to the high false discovery rate of discovery “omics” methods (regardless of technology used), together with a lack of robust methods for biomarker verification in large clinical sample sets (4-7). It is now common for differential analysis of tissue or plasma by multidimensional LC-MS/MS (the workhorse tool for unbiased discovery) to provide confident identification of 1000’s of proteins, 100’s of which can vary 5-fold or more between case and control samples in small discovery studies. In order to access proteins at lower abundance (e.g., sub 500 ng/mL in plasma, levels at which many known protein biomarkers like carcinoembryonic antigen, PSA, neuron specific enolase, and the troponins occur), these studies always employ multidimensional fractionation at the protein and/or peptide level, thus exploding a single patient sample into up to a 100 sub-fractions, each requiring lengthy LC-MS/MS analysis. It is not uncommon for the analysis of a single case/control sample pair to take up to two weeks of on-instrument time. This limits the numbers of samples that can be practically analyzed to typically 10 (or fewer) case vs control comparisons. These numbers are very small relative to the high dimensionality of the proteome (100,000’s or more possible components when posttranslational modifications and other variants are taken into account), and the scale of normal variation in the human population. Thus a very large fraction, possibly exceeding 95% of the protein biomarkers “discovered” in these experiments are false positives arising from biological or technical variability. Clearly discovery “omics” experiments do not lead to biomarkers of immediate clinical utility, but rather produce “candidates” that must be “qualified” and “verified” (6,7).
Until recently verification technologies capable of testing large numbers of protein biomarker “candidates” emerging from discovery “omics” experiments in large (>1,000-2,000) sample sets have not been available. In principal antibody (Ab)-based measurements could be used. However the required immunoassay-grade Ab pairs exist for only a small number of the potential candidate biomarker proteins. Developing a new, clinically deployable immunoassay is very expensive ($100K - $250K per biomarker candidate for a research assay, or $2-4M for an FDA-approvable assay) and time consuming (1-1.5 yrs) which restricts their use to the short list of already highly credentialed candidates. For the large majority of new, unproven candidate biomarkers an intermediate verification technology is required that has shorter assay development timelines, lower assay cost, effective multiplexing of 10-50 candidates, low sample consumption and throughput capable of analyzing 100’s to 1,000’s of samples of serum or plasma with good precision. The goal of this verification is to identify those few candidate protein biomarkers from the initial list of hundreds that are worth advancing to traditional candidate validation studies using assays deployable on a clinically approved analysis platforms.
The core technology that has emerged for candidate biomarker verification is Stable Isotope Dilution (SID) - Multiple Reaction Monitoring (MRM) Mass Spectrometry (8,9), an approach that has been very successful for quantitation of small molecules (e.g., hormones, drugs and their metabolites) in pharmaceutical research and more recently in clinical laboratories. Use of SIDMRM-MS for protein assays is predicated on measurement of “signature” or “proteotypic” tryptic peptides that uniquely and stoichiometrically represent the protein candidates of interest. MRM-based assay development starts with selection of 3-5 peptides per protein (9). Synthetic, stable isotope labeled versions of each peptide are used as internal standards, enabling protein concentration to be measured by comparing the signals from the exogenous labeled and endogenous unlabeled species. Peptide selection is driven by the initial discovery data, as well as additional experiments such as Accurate Inclusion Mass Screening (10) and information available in on-line databases such as GPM (11) and PeptideAtlas (12) identifying peptides that have been observed in other proteomics experiments. Response curves for each peptide in the matrix consisting of trypsin-digested plasma are obtained to evaluate potential interferences and to establish the LOQ and LOD for each peptide. One to two configured assays are produced for any given protein.
SID-MRM-MS assays have several distinguishing features relative to conventional immunoassays. First, the analyte detected in the MS can be characterized with near-absolute structural specificity – something never possible using Ab's alone. This provides a potentially critical quality advantage, especially in cases where immunoassays are subject to interferences. Second, MRM assays can be highly multiplexed such that 20 or more proteins can be measured during a single analysis (8,9), with assay CV's of <10% demonstrated for proteins at 1 ug/mL level or higher in plasma (8). Third, all of these measurements can be done on ca. 100 nL of plasma. Individual immunoassays often consume 10-100 uL of plasma (i.e., 100-1,000 times as much).
Many biomarkers of current clinical importance such as PSA, CEA and the troponins reside in the 10’s of picograms to low nanogram/mL range in plasma. Are SID-MRM-MS methods capable of such sensitivity? Keshishian et al. have recently shown that a combination of abundant protein depletion combined with minimal fractionation of tryptic peptides by strong cation exchange prior to SID-MRM-MS provides LOQs in the 1-20 ng/mL range with CV's of 10-20% at the limits of quantitation for these proteins in plasma (9). However, this extent of sample processing restricts sample throughput substantially compared to immunoassay. Detection of proteins in the mid-to-low pg/mL range is not currently possible using this approach because of current limits to MS sensitivity. Improvements in instrument design on the near horizon may help break this barrier (for example, see 13.14).
Stable Isotope Standards with Capture by Anti Peptide Antibodies (SISCAPA) combines the advantages of specific immunoaffinity enrichment of a target peptide with the structural specificity and quantitative capabilities of SID-MRM-MS (15,16). In this approach anti-peptide antibodies are made against the selected signature tryptic peptides from the proteins of interest. Following digestion of the plasma and addition of known amounts of stable isotope labeled standard peptide, both added and sample-derived versions are specifically enriched and the relative amounts measured by MRM. While the affinity of the Ab for the peptide must be quite good, requirements for selectivity can be relaxed since the mass spectrometer is capable of specifically detecting and quantifying the signature peptides even in the presence of a highly complex background. Recent studies suggest that more than a thousand-fold enrichment can be achieved for plasma digest peptides using this approach (16), and that SISCAPA assays can achieve low ng/ml LOQ in plasma with CV < 20%. In substituting one Ab affinity step at the peptide level for more complex multi-step sample fractionation schemes, SISCAPA improves throughput (e.g., in magnetic bead format) while likely permitting at least 10 assays to be multiplexed.
The paper by Hoofnagle and coworkers in this issue of Clinical Chemistry is another important contribution to the emerging SID-MRM-MS and SISCAPA literature. These authors employ a polyclonal anti-peptide Ab to develop the first SISCAPA assay implemented in a clinical laboratory environment – an assay for serum thyroglobulin, an established tumor marker whose existing immunoassays are plagued by frequent interferences negatively affecting clinical performance (17,18). Low ng/mL LOQs are demonstrated with acceptable assay CVs, consistent with sensitivity and assay CVs reported for other MRM (9) and SISCAPA (15,16) assays, and the results correlate well with current immunoassay results. The work provides a fine example of how MRM and SISCAPA assays can be readily configured for new target proteins, and how these avoid many of the common problems associated with immunoassays when the analyte is in the current detection range of >1 ng/ml in blood. By using peptide (as opposed to protein) immunoaffinity enrichment prior to SID-MRM-MS, the authors avoid potential interferences commonly encountered with endogenous immunoglobulins in immunoassays (such interfering components being digested to non-interacting peptides prior to specific capture of target peptides). However, the attractive notion that SISCAPA assays will be entirely interference-free is probably not correct; while they will likely not suffer from host Ab interference, they can be subject to interference from peptides having nearly the same epitope or the same epitope in a slightly different sequence context that will still be recognized and captured by the Ab. Nevertheless, unlike immunoassay measurements, provided the interfering peptide mass is even a few Da different from that of the target peptide, it will be immediately clear that an interference is present.
Is quantitative mass spectrometry of peptides ready to follow MRM of steroids and immunosuppressants and into the clinical lab? MRM methods coupled with SISCAPA have the potential to produce results of sufficient sensitivity, reproducibility and ruggedness to eventually be adopted into clinical labs. They already have the distinct advantages over ELISA methods of enabling rapid drafting of new assays at relatively low cost while retaining the ability to produce very high quality results. SISCAPA-MRM methods also have unique advantages (relative to ELISA) such as definitive characterization of analyte structure, facile detection of interferences, and ease of multiplexing. This tremendous promise may eventually be realized in the clinical lab if future studies demonstrate good intra- and inter-lab assay reproducibility across a wide range of protein analytes and if assay sensitivity can be further improved. Compelling and repeated demonstration of SISCAPA-MRM capabilities will hopefully lead instrument vendors to develop the fully automated and reliable sample preparation and LC-MS/MS instruments that will be required for routine clinical laboratory use, as well as a full menu of assays for known protein biomarkers. Such developments would spur radical improvements in biomarker verification and validation, and provide a far smoother path from biomarker discovery to clinical implementation than exists today.