High Sensitivity Measurement of Parathyroid Hormone–Related Protein (PTHrP) in Plasma by LC-MS/MS

Abstract

N-terminal sequence of parathyroid hormone–related protein (PTHrP) has close homology to parathyroid hormone (PTH). In health, both PTH and PTHrP participate in calcium regulation and homeostasis, but some of the functions, such as regulation of bone development, teeth eruption, calcium regulation in central nervous system, and calcium regulation during pregnancy and fetal development, are unique to PTHrP. In pathology, PTHrP is involved in activation of the pathways, allowing tumor cells to form bone metastasis. In contemporary clinical practice, measurements of PTHrP are used for diagnosing and management of patients suspected of hypercalcemia of malignancy. We describe high-sensitivity, high-specificity LC-MS/MS method for measurement of PTHrP. Sample preparation in this method is performed as follows: internal standard (¹⁵N labeled PTHrP) is added to plasma samples. PTHrP and the internal standard are enriched from the samples using anti-PTHrP antibody conjugated to magnetic beads. The beads are washed, PTHrP is digested with trypsin, and a PTHrP-specific signature peptide is analyzed using LC-MS/MS. The lower limit of detection, limit of quantitation, and upper limit of linearity of the assay are 0.5, 2, and 600 pmol/L; total imprecision of the method is <10%. Reference intervals for PTHrP established using this method in samples of healthy women and men are <3.4 pmol/L and < 2.3 pmol/L, respectively. The method has acceptable performance for use in clinical diagnostic applications.