Abstract
Pre-eclampsia is a pregnancy-specific disease that complicates 2-8% of all pregnancies. It is associated with serious perinatal and maternal morbidity and mortality. We previously demonstrated that in pre-eclampsia placenta, calcyclin expression is significantly higher compared to controls. The sensitivity of this analysis was improved by Selective Reaction Monitoring (SRM) using a Xevo TQ-S mass spectrometer enabling the quantitation of calcyclin at low attomole levels in trophoblast cells. Next, a method was developed to quantify calcyclin in digested sera to evaluate calcyclin as a potential serum biomarker. An SRM assay was developed for quantitative measurements of calcyclin in serum using stable isotopic labeled peptides. We compared three sample preparation methods, digestion without any further sample treatment, digestion of affinity depleted serum and off-line fractionation of digested sera with a Luna 5 μm, 150 x 2 mm SCX column. Samples were measured using a nanoACQUITY LC system equipped with a BEH130 C18, 1.7 μm, 75 μm x 250 mm column connected to a Xevo TQ-S triple quadrupole mass spectrometer. For all methods, linearity and limit of detection (LOD) of the assay were determined. Endogenous levels of calcyclin were detectable in undepleted control sera but quantitation was not feasible due to high biological background and low signal intensity. For depleted and SCX fractionated serum samples, endogenous levels could be measured. We obtained LODs of 5 and 2.5 ng/ml serum, respectively, equivalent to 10 and 5 attomole of calcyclin peptides on column. Best results were obtained using SCX fractionation of the serum samples; quantitation could be performed for both selected peptides without interference. The average observed concentration of calcyclin in control serum samples was 35 ng/ml. We have developed and validated a method that is sufficiently sensitive and robust to quantify endogenous levels of calcyclin in serum and trophoblast cells of placental tissue.