Targeted peptide measurements in biology and medicine: Best practices for mass spectrometry-based assay development using a fit-for-purpose approach

Steven A. Carr*, Susan E. Abbatiello, Bradley L. Ackermann, Christoph Borchers, Bruno Domon, Eric W. Deutsch, Russell P. Grant, Andrew N. Hoofnagle, Ruth Hüttenhain, John M. Koomen, Daniel C. Liebler, Tao Liu, Brendan MacLean, D. R. Mani, Elizabeth Mansfield, Hendrik Neubert, Amanda G. Paulovich, Lukas Reiter, Olga Vitek, Ruedi AebersoldLeigh Anderson, Robert Bethem, Josip Blonder, Emily Boja, Julianne Botelho, Michael Boyne, Ralph A. Bradshaw, Alma L. Burlingame, Daniel Chan, Hasmik Keshishian, Eric Kuhn, Christopher Kinsinger, Jerry S.H. Lee, Sang Won Lee, Robert Moritz, Juan Oses-Prieto, Nader Rifai, James Ritchie, Henry Rodriguez, Pothur R. Srinivas, R. Reid Townsend, Jennifer Van Eyk, Gordon Whiteley, Arun Wiita, Susan Weintraub

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

442 Scopus citations


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 and the experimental evidence needed to establish that the assays they develop work as intended and are achieving the required levels of performance. Using this "fit-for-purpose" approach, the group defined three tiers of assays distinguished by their performance and extent of analytical characterization. Computational and statistical tools useful for the analysis of targeted MS results were described. Participants also detailed the information that authors need to provide in their manuscripts to enable reviewers and readers to clearly understand what procedures were performed and to evaluate the reliability of the peptide or protein quantification measurements reported. This paper presents a summary of the meeting and recommendations.

Original languageEnglish
Pages (from-to)907-917
Number of pages11
JournalMolecular and Cellular Proteomics
Issue number3
StatePublished - Mar 2014
Externally publishedYes


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