scope:

This guideline provides broad recommendations for appropriately developing and validating quantitative protein and peptide assays for clinical applications using electrospray liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques. C64 is practically focused and includes workflow overviews and experimental strategies for developing and validating quantitative assays for soluble proteins and peptides in biofluids (eg, serum, saliva, urine). It covers complex analyses, including measurement of proteins with post-translational modifications (PTMs). Although there are a diverse array of ionization modes and associated mass analyzers (eg, matrix-assisted laser desorption/ionization time-of-flight [MALDI-TOF] mass spectrometry [MS]), this guideline focuses on liquid chromatography (LC) and electrospray ionization (ESI) coupled with tandem mass spectrometry (MS/MS) because of the wide availability and proven utility of this method.

A protein or peptide associated with a medical diagnosis or clinical outcome may exist in vivo as a single specified molecular composition or as a complex collection of related proteoforms that differ in molecular composition. Given the heterogenous nature of proteins and the desirability of facilitating results standardization among different assays, the need to appropriately define the measurand is a key difference between small-molecule analysis and protein analysis. In order to design a suitable workflow for measurand assessment, the assay developer needs to consider analyte properties, enrichment and fractionation strategy, and instrument performance characteristics. Subsequently, calibrators and internal standards (IS) are selected based on the chosen workflow and a precisely defined measurand. At the beginning of method development, performance criteria guide the conception and refinement of the path of workflow. Following this iterative process, the developer eventually prepares a candidate method sufficiently robust to pass validation. Finally, rigorous validation studies are performed to demonstrate suitability for routine clinical use.

The intended users of this guideline are medical, research, and public health laboratories; in vitro diagnostic instrument manufacturers; and regulatory and accreditation organizations.

Tissues and other nonbiofluid specimens are not within this guideline's scope. Enzyme activity assays are also considered out of scope, as are detailed discussions of software tools and data processing algorithms used for in silico analysis of protein and peptide sequences.