Proteoform profiling of peripheral blood serum proteins from pregnant women provides a molecular IUGR signature

Abstract

Intrauterine growth restriction (IUGR) is an important cause of perinatal morbidity and mortality and contributes substantially to medically indicated preterm birth; preventing fetal death. Molecular profiling of the mothers' peripheral blood was desired to monitor the health conditions of the fetuses. To develop such a minimally invasive assay, we applied a protein affinity fractionation method to peripheral blood serum samples from pregnant women belonging to either the IUGR or to the control group. Proof-of-principle was shown by relative quantitation analysis of mixtures of intact proteoforms using MALDI-ToF mass spectrometry. The two best differentiating proteins and proteoforms, respectively, were apolipoprotein C-II and apolipoprotein C-III₀. Together with three robustly expressed protein proteoforms proapolipoprotein C-II, apolipoprotein C-III₁, and apolipoprotein C-III₂, which served as landmarks for relative quantitation analysis, they constituted the maternal IUGR proteome signature. Separation confidence of our IUGR proteoform signature reached a sensitivity of 0.73 and a specificity of 0.87 with an area under curve of 0.86 in receiver operator characteristics.

Significance: Identification of IUGR newborns in the case room is required as children are severely diseased and need specialized care during infancy. Yet, at time of birth there is no readily applicable clinical test available. Hence, a molecular profiling assay is highly desired. It needs to be mentioned that current clinical definitions and recommendations for IUGR are unfortunately misleading and are not universally applicable. The most commonly adopted definition is an abdominal circumference (AC) or estimated fetal weight measurement <10th percentile. Although both, the American College of Obstetricians and Gynecologists (ACOG) and the Royal College of Obstetricians and Gynecologists (RCOG) agree that at this cut-off the risk of perinatal morbidity and mortality increases, this definition does not take into account the individualized growth potential of each fetus. In particular its sole use fails to identify larger fetuses that have not achieved their growth potential and may be at risk of adverse outcomes. Also, this definition, when solely applied, will result in the misdiagnosis of IUGR for some constitutionally small fetuses. It needs to be pointed out that the above mentioned criteria can only be determined during pregnancy in case mothers report from early on during pregnancy. We have developed a test that relies on mass spectrometric analysis of the mother's serum protein composition (IUGR signature) which can be determined just ahead of delivery and at date of delivery, respectively using a minimal invasive blood sampling approach. With this manuscript we describe the use of a mass spectrometric profiling method of 30 peripheral blood samples from pregnant women prior to giving birth of either unsuspicious newborns or IUGR-affected infants. We report for the first time that maternal blood sample analysis via affinity mass spectrometry differentiates IUGR infants from controls with high confidence.

Keywords: Affinity - mass spectrometry; IUGR; MALDI-ToF MS; Multiparametric profiling; Peripheral venous blood analysis; Proteome signature.