Rainer Bischoff
University of Groningen
| Short Abstract The quantification of proteins in complex biological samples is central to many areas of research, as well as to industrial and clinical applications. Especially the clinical chemistry laboratory uses quantitative protein assays on a daily basis to assist in medical decision making, for example, in defining the best therapy for a given disease or in disease diagnosis. While quantitative protein assays have relied and rely to a major part on ligand binding assays (LBAs) and in particular on immunoassays, we experience the advent of liquid chromatography – mass spectrometry (LC-MS) assays as an alternative or complement to LBAs. In this lecture I will delineate the advantages and shortcomings of LBAs and LC-MS assays with the goal of showing that there is no single approach that can answer all questions. I will notably address the point that proteins do not exist as single molecular entities in vivo and that we must therefore refer to proteins as families. |
Long Abstract
The quantification of proteins in complex biological samples is central to many areas of research, as well as to industrial and clinical applications. Especially the clinical chemistry laboratory uses quantitative protein assays on a daily basis to assist in medical decision making, for example, in defining the best therapy for a given disease or in disease diagnosis. While quantitative protein assays have relied and rely to a major part on ligand binding assays (LBAs) and in particular on immunoassays, we experience the advent of liquid chromatography – mass spectrometry (LC-MS) assays as an alternative or complement to LBAs. In this lecture I will delineate the advantages and shortcomings of LBAs and LC-MS assays with the goal of showing that there is no single approach that can answer all questions. I will notably address the point that proteins do not exist as single molecular entities in vivo and that we must therefore refer to proteins as families. The question then arises, what is the concentration of 'a protein' in blood or another body fluid? Interpreting the results of quantitative protein determinations must take the measurement principle into account, be it an LBA or an LC-MS assay, since we only measure a small portion of any given protein (e.g. an epitope or a signature peptide). It is, thus, not totally surprising that results between LBAs and LC-MS may differ, since each method may measure a different, partially overlapping part of a given protein family.1
To demonstrate the possibilities of LC-MS assays, I will refer to a number of examples. The first example is from the area of biopharmaceuticals, where we studied the in vivo biotransformation of the monoclonal antibody Trastuzumab, which is used in a subset of breast cancer patients. In this work we show that a critical Asn residue in the receptor binding site deamidates and that the extend of deamidation differs between patients.2 It is noteworthy that concentration measurements in plasma by a targeted LC-MS assay and a widely used immunoassay (ELISA) differ by as much as a factor of 2 depending on the level of deamidation. The following example will address another important issue that is related to the use of biopharmaceuticals as therapeutic agents, namely the development of anti-drug antibodies (ADAs). Patients taking protein pharmaceuticals over a long time period, possibly lifelong, run the risk of developing an immune response, which hampers the efficacy of the biopharmaceutical drug. We studied this using LC-MS in the case of enzyme replacement therapy with alpha-glucosidase in Pompe Disease patients showing that ADAs can be measured accurately and precisely.3 The final examples focus on the development of high-sensitivity (LC)-MS methods for biomarkers at the sub-ng/mL level.4-6
References
(1) Bults, P.; Van De Merbel, N. C.; Bischoff, R. Quantification of biopharmaceuticals and biomarkers in complex biological matrices: A comparison of liquid chromatography coupled to tandem mass spectrometry and ligand binding assays. Expert Review of Proteomics 2015, 12, 355-374.
(2) Bults, P.; Bischoff, R.; Bakker, H.; Gietema, J. A.; van de Merbel, N. C. LC-MS/MS-based monitoring of in vivo protein biotransformation: quantitative determination of trastuzumab and its deamidation products in human plasma. Analytical Chemistry 2016, 88, 1871-1877.
(3) Bronsema, K. J.; Bischoff, R.; Pijnappel, W. W. M. P.; van der Ploeg, A. T.; van de Merbel, N. C. Absolute Quantification of the Total and Antidrug Antibody-Bound Concentrations of Recombinant Human α-Glucosidase in Human Plasma Using Protein G Extraction and LC-MS/MS. Analytical Chemistry 2015, 87, 4394-4401.
(4) Klont, F.; ten Hacken, N. H. T.; Horvatovich, P.; Bakker, S. J. L.; Bischoff, R. Assuring Consistent Performance of an Insulin-Like Growth Factor 1 MALDImmunoassay by Monitoring Measurement Quality Indicators. Analytical Chemistry 2017, 10.1021/acs.analchem.7b01125
(5) Wilffert, D.; Asselman, A.; Donzelli, R.; Hermans, J.; Govorukhina, N.; Quax, W. J.; van de Merbel, N. C.; Bischoff, R. Highly sensitive antibody-free microLC-MS/MS quantification of rhTRAIL in serum. Bioanalysis 2016, 8, 881-890.
(6) Wilffert, D.; Reis, C. R.; Hermans, J.; Govorukhina, N.; Tomar, T.; de Jong, S.; Quax, W. J.; van de Merbel, N. C.; Bischoff, R. Antibody-free LC-MS/MS quantification of rhTRAIL in human and mouse serum. Analytical Chemistry 2013, 85, 10754-10760.
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