Tremendous progress has been made in using multiple reaction monitoring MS (MRM-MS) for verification of clinical biomarkers, for which it is widely used and accepted. Targeted proteomics, in which a subset of candidate biomarkers are detected and measured, using MRM-MS enables multiplex, high-throughput quantification from a few up to hundreds of proteins per sample, reducing the long list of potential biomarkers to the most promising proteins. Major developments in MRM-MS for biomarker verification include reduced variability, new research resources and increased sensitivity. The current challenge is validating these potential biomarkers for clinical use, including FDA clearance. Thus, while MRM-MS’s use for clinical biomarker verification is well proven, the transition of MRM-MS assays to diagnostic applications remains a complex and continually evolving area.

MRM-MS detects and quantifies a sample’s proteins using surrogate peptides. Using a triple quadrupole MS, the relevant ions in a sample are selected, filtered and measured. Stable isotope-labeled (SIL) peptides serve as a standard, enabling absolute quantitation. The advantages of MRM-MS over immunoassays for biomarker verification include multiplexing capability, higher-throughput and shorter development time. In addition, MRM-MS can detect post-translational modification of proteins.

Among the programs that have advanced the use of MRM-MS for clinical biomarker verification is the NCI’s Clinical Proteomic Tumor Analysis Consortium (CPTAC). Established in 2006, CPTAC’s initial focus included the development of proteomics-based technologies for clinical biomarker applications. In particular, CPTAC worked to increase the standardization and rigor of MRM-MS experiments for verifying potential clinical biomarkers. For MRM-MS, CPTAC’s accomplishments include demonstrating reproducibility and interlab transferability; development of public resources, such as experimental guidelines and high quality reagents; and platform validation. CPTAC’s efforts also addressed technical disadvantages of MRM-MS, including the time and effort required to develop MRM-MS assays. Last year, CPTAC launched the Assay Portal, a repository of well-characterized MRM-MS assays.

CPTAC’s output has also included workshops and publications to advance MRM-MS standardization and validation, including interlaboratory studies and a mock 501(k) submission. For example, a 2013 workshop held by CPTAC and National Heart, Lung, and Blood Institute Proteomics Centers resulted in the publication of a paper by Steven A. Carr, PhD, et al., in March 2014 in Molecular & Cellular Proteomics entitled “Targeted Peptide Measurements in Biology and Medicine: Best Practices for Mass Spectrometry-based Assay Development Using a Fit-for-Purpose Approach.” The paper describes the development of three tiers of targeted MRM-MS assays and the appropriate analytical validation required for each.

The development and commercialization of standards have also been a key advancement for MRM experimental reproducibility and quality control, according to Christoph Borchers, PhD, director of the University of Victoria’s Genome BC Proteomics Centre and CSO of MRM Proteomics, a provider of SIS peptide kits and services for LC-MRM/MS. Earlier this year, MRM named Cambridge Isotope Laboratories as the exclusive distributor of PeptiQuant Kits, including the Biomarker Assessment Kit for quantitating 76 proteins associated with various diseases in one experiment. He told IBO that new kits will be introduced later this month at the Human Proteome Organization’s conference.

Concurrent with CPTAC’s efforts to standardize and validate MRM approaches for biomarker verification, new sample preparation techniques have been developed and widely adopted to further increase the sensitivity and throughput of MRM, particularly for low-abundance proteins. Using immuno-enrichment strategies, the analytes per sample can be measured in the pg/mL range in blood or plasma. Immuno-enrichment utilizes immunoaffinity to pull out target proteins, lowering potential interference from other sample components.

Among the most prominent immuno-enrichment techniques is SISCAPA (stable isotope standards and capture by antipeptide antibodies), which utilizes peptide enrichment using antibodies with SIL proteins. The technique is robust and can be automated using standard laboratory automation platforms. Used for biomarker candidate verification, it has also been commercialized as clinical diagnostic LDT single-plex assays for thyroglobulin testing for posttreatment monitoring of thyroid carcinoma.

As Leigh Anderson, PhD, CSO of SISCAPA Assay Technologies and inventor of SISCAPA, told IBO, SISCAPA was designed to increase the sensitivity and throughput of MS analysis for biomarker verification. “SISCAPA is a method, typically using an antibody against a specific peptide, to enrich that peptide out of a really complicated sample. That has two effects: first, you can pull this peptide analyte out of a much larger amount of sample than you could ever inject into a mass spectrometer, and so you can improve the sensitivity at least a thousand fold by just accessing more of the analyte in a larger amount of sample,” he explained. “And, then, secondarily, it effectively purifies the analyte that you are going to be measuring. So, instead of injecting an incredibly complicated and dirty sample into the mass spec or LC/MS system, you are injecting effectively pure analyte. That means, of course, that the interferences are drastically reduced and the need for chromatography itself is drastically reduced.” SISCAPA is typically used with low-flow LC/MS, according to him. However, it can also be used with MALDI-TOF and Agilent Technologies’ RapidFire flow-injection system. “We typically run anywhere from a 6-plex to a 22-plex on a regular basis, and people have run on several occasions, put together 50-plex panels with SISCAPA.”

Dr. Anderson’s company sells SISCAPA reagents, such as antipeptide antibodies, SIS-labeled proteins and unlabeled proteins, as well as licenses. It is also an Agilent value-added reseller. To promote and advance the use of SISCAPA for clinical diagnostics, SISCAPA Assay Technologies is developing assays. “So we are in the process of building a large menu of biomarker candidate assays with SISCAPA reagents,” he noted. The company is also creating SISCAPA assays for each of the 105 proteins in FDA-approved immunoassays. “We’re about 25% of the way through that menu. In another couple of years, we’ll have the entire clinical menu of existing protein tests.”

Dr. Anderson believes it is important that the instrument platform on which such verification studies are performed are the same as the one eventually used for the diagnostic test. “There is a huge opportunity open to mass spectrometry uniquely in the clinical laboratory for this reason: if it turns out to be possible with mass spectrometry to do the verification/validation and the clinical implementation on the same platform or very similar platform, then that effectively takes five years out of the conventional development process to get a biomarker into the clinic,” he told IBO. The current uses of FDA-registered Class 1 triple quadrupole MS for diagnostic applications, such as neonatal screening, vitamin D testing and therapeutic drug monitoring of immunosuppressant drugs, have put MS in some clinical labs, but it is not widely used, and such tests measure small molecules rather than proteins.

Both Drs. Borchers and Anderson agreed that large-scale validation studies are the next step for advancing MRM-MS-based tests for biomarkers toward diagnostic applications. However, both acknowledged that there are few incentives for academic researchers to conduct such studies, and it is a cost prohibitive undertaking for private companies in many cases. Another issue is getting large enough sample sets to conduct such testing.

In addition to large-scale testing, questions remain about the requirements for analytical and clinical validation for multiplex MRM-MS assay, a new category for FDA clearance and one far different from traditional protein immunoassays. For example, validation of each analyte in a multiplex panel may be required. An example of the regulatory expectations for such tests is provided in the March 2014 Molecular & Cellular Proteomics article describing three tiers of targeted proteomic assays; Tier 1 describes tests designed for clinical bioanalysis and diagnostic tests. Tier 1 tests require a high degree of analytical validation, labeled internal standards for every analyte, reference standards, high specificity, precision of less than 20%–25% CV and high repeatability.

The requirements for triple quadrupole MS instrumentation for FDA-approved multiplex MRM-MS assays remain undetermined, including the extent of regulatory compliance. As Emily S. Boja, PhD, Program manager for the NCI’s Office of Cancer Clinical Proteomics Research, et al. wrote in an article entitled, “Analytical Validation Considerations of Multiplex Mass-Spectrometry-Based Proteomic Platforms for Measuring Protein Biomarkers” in the December 2014 issue of the Journal of Proteome Research, “Regulatory evaluation of instrument platforms should encompass all components and accessories (e.g., nanoflow columns, trap columns, silica tubings, electrospray source, and predictive software if needed). Even the slightest changes to a predicate would have to be reexamined by the assay sponsor prior to submissions to ensure the safety and effectiveness of the modified platform.”