The development and use of isotope dilution mass spectrometry methods for the quantification of target proteins in certified reference materials.

Originally developed for small molecules in the late 1970s and early 1980s, isotope dilution mass spectrometry (IDMS) also has become the "go to" method of quantification for those wishing to characterize the mass fraction or amount of substance content of proteins in matrix-based certified reference materials (CRMs). While instrumental factors can impact the accuracy of IDMS methods, the use of double IDMS, higher order approaches, and "exact matching" have done a lot to improve the accuracy of the results. This review shows the development of IDMS for the quantification of proteins from being a method only used for value assignment of primary calibrators using isotope-labelled peptides as internal standards to a method for quantification of proteins in complex biological matrices such as serum and blood using isotope-labelled recombinant intact proteins as internal standards. It will discuss the challenges and limitations of currently used organic IDMS peptide-based approaches regarding the definition of the measurands, equilibration of sample and internal standard, digestion efficiency, and stability and suitability of the internal standards. This paper reviews the steps taken to assess the accuracy of the peptide- and protein-based IDMS methods used for the characterization of CRMs and the requirement to ensure comparability in laboratory diagnostics down to the patient level. Furthermore, it will give an outlook on future challenges, such as including structural and protein activity information in the results.