Targeted Peptide Measurements in Biology and Medicine: Best Practices for Mass Spectrometry-based Assay Development Using a Fit-for-Purpose Approach

Abstract

Adoption of targeted mass spectrometry (MS) approaches such as multiple reaction monitoring (MRM) to study biological and biomedical questions is well underway in the proteomics community. Successful application depends on the ability to generate reliable assays that uniquely and confidently identify target peptides in a sample. Unfortunately, there is a wide range of criteria being applied to say that an assay has been successfully developed. There is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated. Publications describing targeted MS assays for peptides frequently do not contain sufficient information for readers to establish confidence that the tests work as intended or to be able to apply the tests described in their own labs. Guidance must be developed so that targeted MS assays with established performance can be made widely distributed and applied by many labs worldwide. To begin to address the problems and their solutions, a workshop was held at the National Institutes of Health with representatives from the multiple communities developing and employing targeted MS assays. Participants discussed the analytical goals of their experiments From the ‡Broad Institute of MIT and Harvard, Cambridge, Massachusetts; §Eli

Targeted Proteomics for Multiplexed Verification of Markers of Colorectal Tumorigenesis

“…In the present study, we developed targeted SRM assays (27) and used them to profile the expression of these candidate protein markers in a large, independent group of colorectal tumors, precancerous and invasive. The results revealed potential markers that significantly distinguished CRC tumors from healthy tissues, showed substantial correlation with clinical parameters and are apparently detectable in human plasma.…”

“…Enzyme-linked immunosorbent assays (ELISAs) are still the preferred methods for preclinical verification of candidate biomarkers, but the antibodies used for these assays are characterized by high specificity requirements and elevated production costs, which render them less than ideal for testing large numbers of tumor biomarkers (19,22). Targeted proteomic techniques have been proposed as standard complementary methods for the clinical verification and validation of candidate disease biomarkers, and these methods are being used in an increasing number of clinical proteomic-based cancer studies (19,(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33).…”

Applications of targeted proteomics in systems biology and translational medicine

“…The selection of a peptide quantification strategy should follow a fit‐for‐purpose approach to achieve the right level of specificity, precision, and quantitative accuracy as described recently in a three‐tiered system in Carr and colleagues 37. This system provides clear guidance with respect to the extent of analytical validation required for each major application type, termed Tier 1 to 3 in the publication 37.…”

“…The selection of a peptide quantification strategy should follow a fit‐for‐purpose approach to achieve the right level of specificity, precision, and quantitative accuracy as described recently in a three‐tiered system in Carr and colleagues 37. This system provides clear guidance with respect to the extent of analytical validation required for each major application type, termed Tier 1 to 3 in the publication 37. In this system, a “labeled internal standard” refers to the use of a consistent spike‐in for relative quantitation and most commonly consists of heavy‐labeled peptides, while a “reference standard” is as close as possible to the native protein in the sample and subject to all the same sample preparation steps, its absolute abundance is known, and calibration curves of dilutions can be established.…”

“…They define: (i) candidate discovery; (ii) qualification; (iii) verification; (iv) research assay optimization; (v) biomarker validation; (vi) commercialization 72. As mentioned above, peptide quantification strategy should follow a fit‐for‐purpose approach to achieve the right level of specificity, precision, and quantitative accuracy as described in a three‐tiered system in Carr and colleagues 37. Further, findings of promising biomarkers or signatures from preclinical studies should be followed by clinical testing.…”