Mass spectrometry (MS) is revolutionizing the way that biomarkers are detected and quantified, with proven application in clinical laboratories in toxicology, endocrinology, and biochemical genetics. The detection of parathyroid hormone, (PTH) in human blood is a new case in point.

PTH Testing History

Parathyroid hormone is an 84 amino acid protein (PTH1-84) secreted by the parathyroid glands. The hormone circulates in blood, has a very short half-life and plays an important physiological role in regulating calcium metabolism. PTH is measured in patients with renal impairment, hyper- and hypocalcemia and hyper-parathyroidism. Accurate PTH measurement is crucial to ensure that these patients are treated appropriately and inaccurate test results can lead to poor outcomes.

Laboratories have been measuring PTH for more than 50 years, first with radioimmunoassays and then with several generations of immunoassay. A key challenge of first generation PTH assays included cross-reactivity with circulating, truncated PTH fragments. To avoid cross-reactivity, scientists developed second-generation immunoassays, which rely on two different antibodies to trap and detect the full-length protein. Even though these methods were superior to the first-generation assays, interference from an almost full length putative PTH fragment, PTH7-84, was reported, although no direct evidence for its existence was provided. As a result of these studies, third-generation intact PTH immunoassays were developed using an antibody that recognized only the N-terminal amino acids 1-4 and therefore should avoid cross-reactivity with the putative PTH7-84 interfering fragment.

PTH Detection Challenges

Despite the new methods, inaccuracy is still an issue. In summary, there are three key challenges measuring PTH levels with any assay. First, as mentioned above, a range of truncated fragments exist in addition to full-length PTH1-84 and these fragments are elevated in patients with renal impairment. PTH fragments can interfere with immunoassays since capture antibodies are typically not specific enough to distinguish full length PTH from fragments that may differ by only a few amino acids. Second, the different PTH fragments have not been fully characterized in terms of their clinical relevance and therefore, it is quite possible that commercial tests do not detect and quantify the most important species. Third, although the FDA has approved a number of immunoassays, method-related differences have led to poor comparability between commercial immunoassays currently used to measure PTH and this variability is likely due to lack of antibody specificity. Variability in PTH testing hampers the implementation of clinical guidelines and may increase harm to patients, especially in situations where longitudinal measurements are necessary. The problem is so acute, that physicians often recommend that patients only receive testing in one laboratory facility.

Mass Spectrometry Addresses Sensitivity and Specificity Challenges in PTH Testing

In recent years, researchers have used immunoaffinity enrichment coupled to mass spectrometry (combining the selectivity of immunoaffinity extraction with the specificity of MS detection) to more completely detect, identify and quantify full length PTH and fragments from renal patient plasma.

In 2010, a research team used a previously published immunoaffinity enrichment protocol (MSIA) to selectively extract PTH and low abundance PTH fragments from plasma. They used a full scan MALDI-TOF MS analysis to identify PTH fragments with truncated N and C termini. This method distinguished the full-length PTH1-84 protein and five novel variants in addition to five N-terminally truncated previously identified variants.

Figure Legend:  Top panel:(A), N-terminally truncated PTH variants identified previously. (B), Variants added to map during this study by use of full-scan MSIA (MALDI-TOF MS) (C), Conserved and truncated tryptic fragments chosen for SRM-MSIA.  Bottom panel: MSIA (MALDI-TOF MS) spectra representative of the 12 samples from patients with renal failure (blue) and the 12 from healthy controls (red).  (Credit: Lopez MF1, Rezai T, Sarracino DA, Prakash A, Krastins B, Athanas M, Singh RJ, Barnidge DR, Oran P, Borges C, Nelson RW. Selected reaction monitoring-mass spectrometric immunoassay responsive to parathyroid hormone and related variants. Clin Chem. 2010 Feb;56(2):281-90.)

Subsequently, the researchers developed a specific and fully quantitative, multiplexed assay for full-length PTH and its truncated variants by coupling the MSIA enrichment to selected reaction monitoring (SRM) detection on a triple-quadrupole mass spectrometer. The results demonstrated that all 10 PTH variants were more abundant in samples from renal patients as compared to healthy controls, implying potential clinical significance. Of note, they did not detect the putative PTH7-84 fragment in any samples.

In a later study, a team at King’s College Hospital, London used the same method but included additional SRM transitions to identify phosphorylated PTH variants in clinical samples from patients with renal impairment. Although the biological activity of the phosphorylated species has not yet been defined, they may be clinically important. Interestingly, the Kings College researchers as well as other research teams  have been unable to detect the putative PTH7-84 fragment in any samples, suggesting it may not exist and again highlighting the power of mass spectrometry to accurately identify proteins and protein fragments directly at the sequence level.

The research studies discussed above demonstrate that renal patient blood samples contain many more PTH isoforms than previously thought, including unanticipated post-translational modifications. Taken together, these findings add to the complexity of our understanding of PTH biology and offer insight into why so few immunoassay-based biomarker discoveries have led to successful therapeutics since it appears that antibodies may bind multiple forms of proteins with differing clinical relevance.

PTH Test Harmonization

In 2009, the World Health Organization created and distributed an International Standard for Parathyroid Hormone 1-84 for the calibration of immunoassays and the diagnosis of calcium metabolism disorders . A PTH standardization meeting held in September 2010 led to short- and long-term recommendations for PTH test result comparability. They also established an International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) working group for PTH (WG-PTH 7). Unfortunately, efforts to harmonize immunoassay-based tests that fundamentally lack the specificity to identify molecular isoforms are not likely to succeed.

Improving Results in Clinical Applications with MS

MS offers a more precise way to characterize molecules of interest. Because MS can identify proteins and peptides at the sequence level, it provides the selectivity to detect and measure slightly different isoforms of the same analyte and accurately reveals molecular variations even for low-abundance, short half-life molecules like PTH.

In summary, the fact that MS can unambiguously detect molecular variations on a micro scale—including post-translational modifications—should lead to increased precision for diagnosis, prognosis and discovery of therapeutic targets. Efforts should be taken to fast track MS in clinical applications. Also, test harmonization efforts should focus on MS methods, specifically in the case of PTH and more generally for molecules of interest in disease causation, progression and prognosis.