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Abstract
The quantification of proteins (biopharmaceuticals or biomarkers) in complex biological samples such as blood plasma requires exquisite sensitivity and selectivity, as all biological matrices contain myriads of proteins that are all made of the same 20 proteinogenic amino acids, notwithstanding post-translational modifications. This review describes and compares the two main approaches, namely, ligand binding assays (LBAs) and liquid chromatography coupled to tandem mass spectrometry in the selected reaction monitoring (SRM) mode. While LBAs remain the most widely used approach, SRM assays are gaining interest due to their generally better analytical performance (precision and accuracy) and their capacity for multiplex analyses. This article focuses on the possible reasons for the discrepancies between results obtained by LBAs and SRM assays.
Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
Proteins occur as groups of molecular entities (called protein species or protein forms) rather than single molecular species. This is a fundamental difference to small molecules, which makes their analytics much more challenging.
Different protein species have different physicochemical properties and may thus respond differently to different analytical assay techniques. This is one of the reasons why Selected Reaction Monitoring (SRM) assays and ligand binding assays (LBAs) often given different results.
Proteins may be modified by post-translational modifications (PTMs). There are more than 350 PTMs reported in the literature, making the proteome a much more complex entity than the genome. PTMs may affect SRM assays and LBAs depending on the chosen signature peptide and the recognition element for an antibody.
The differences between the results obtained by SRM assays and LBAs are caused by the interferences of anti-protein antibodies or other molecules binding to a given protein with the assay. For example, an anti-protein antibody may interfere with the binding of a capturing antibody used in an LBA.
SRM assays have the capacity to measure multiple analytes (e.g., biomarkers) in a single run. This opens the field of defining molecular pattern rather than single-molecule diagnostic assays, an area that is likely to thrive in the future.