Analysis of β-nerve growth factor and its precursor during human pregnancy by immunoaffinity-liquid chromatography tandem mass spectrometry

Abstract

β-Nerve growth factor (NGF) is a neurotrophin that plays a critical role in fetal development during gestation. ProNGF is the precursor form of NGF with a distinct biological profile. In order to investigate the role of NGF and proNGF in pregnant human females, a sensitive and selective immunoaffinity liquid chromatography-tandem mass spectrometry assay was developed and qualified to simultaneously measure the levels of total NGF (tNGF; sum of mature and proNGF) and proNGF using full and relative quantification strategies, respectively. The assay was used to determine serum tNGF and proNGF levels in the three gestational trimesters of pregnancy and in non-pregnant female controls. Mean tNGF ± SD were 44.6 ± 12.3, 42.6 ± 9.3, 65.4 ± 17.6 and 77.0 ± 17.8 pg/mL for non-pregnant, first, second, and third trimesters, respectively, demonstrating no significant increase in circulating tNGF between the control and the first trimester, and a moderate yet significant 1.7-fold increase through gestation. proNGF levels during the first trimester were unchanged compared to control. In contrast to tNGF, however, proNGF levels during gestation remained stable without significant changes. The development of this sensitive, novel immunoaffinity duplexed assay for both tNGF and proNGF is expected to enable further elucidation of the roles these neurotrophins play in human pregnancy as well as other models.

Figure 1

(A) Representative calibration curve for tNGF. (B) tNGF in pregnancy samples (1) control, (2) 1st trimester, (3) 2nd trimester, (4) 3rd trimester. (C) proNGF (1) control, (2) 1st trimester, (3) 2nd trimester, (4) 3rd trimester.

Figure 2

Mean proNGF L:H PAR (light: heavy peak area ratio) for all proNGF QCs generated by admixing proQCL with proQCH showing a 2.37-fold increase in proNGF L:H PAR from proQCL to proQCH.

Figure 3

(A) tNGF and (B) proNGF levels in non-pregnancy and pregnancy cohorts broken down by trimester. Shown below each plot are the mean and standard deviation (SD) for each group as well as the p value as compared to the control (non-pregnant) group. Lines indicate the mean of each group.