From Method-Defined Signals to Reference Measurement Procedures: Two Decades of Mass Spectrometry-Based ProGRP Quantification.

Pro-gastrin-releasing peptide (ProGRP) is a clinically established biomarker in small cell lung cancer (SCLC) and other neuroendocrine neoplasms (NENs). Despite widespread clinical use, ProGRP measurement is challenged by low circulating concentrations and proteoform heterogeneity arising from alternative precursor processing. Current immunoassay-based methods generate operationally defined signals anchored to antibody-epitope interactions rather than chemically explicit molecular quantities, limiting result comparability across platforms. Mass spectrometry (MS) addresses these limitations through sequence-defined quantification, with the capacity to resolve sequence-distinct isoforms and, at the intact-protein level, individual proteoforms. In this Perspective, we examine two decades of MS-based ProGRP quantification as a case study for quantitative proteomics and measurement science. We trace the evolution from single-quadrupole workflows to isotope dilution strategies enabling increasingly selective and metrologically robust measurement, showing that performance gains were driven primarily by selectivity engineering and internal standardization rather than by mass analyzer advances. This trajectory progressively exposes a fundamental comparability problem: immunoassays and MS-based methods do not measure the same quantities. Resolving this discordance requires an explicit metrological framework encompassing measurand definition, traceability architecture, and reference measurement procedures (RMPs) based on protein-level isotope dilution MS. ProGRP serves as an instructive model for harmonizing quantitative protein biomarker measurements in clinical proteomics.