Amniocytes are free-flowing fetal cells present in amniotic fluid. While they are used routinely for prenatal diagnostic purposes, a full characterization of these amniotic cells has not been done. These fetal cells originate from all three germ layers of the fetus,1 and cells resulting from Trisomy 21 are expected to be distinctly different from a chromosomally typical fetus. To better understand the composition of these cells on a proteomic level, the Diamandis group performed a quantitative analysis of amniocytes. In particular, they predicted proteins in cells with Trisomy 21 would show a deregulated molecular pathway when compared with chromosomally normal amniocytes.2
Amniocyte cultures were grown in serum-free media and control pair mixtures were created with equal amounts of proteins from two individuals at the same gestational age. Pairs were established containing chromosomically typical proteins as well as typical proteins mixed with Trisomy 21 proteins. Proteins were digested with trypsin for mass spectrometry. The Diamandis group employed stable isotope labeling of amino acids in cell culture (SILAC), followed by tandem mass spectrometry on an LTQ Orbitrap XL mass spectrometer (Thermo Scientific) coupled to an Agilent 1100 HPLC.
Additional amniocyte samples were collected at 15 to 18 weeks of gestation and cultured for the second phase of analysis. Phase two consisted of selected reaction monitoring (SRM) of candidate proteins separated via liquid chromatography and analyzed via tandem mass spectroscopy on a TSQ Vantage (Thermo Scientific) coupled to a nanoelectrospray ionization source (nano-ESI; Proxeon A/S). XCalibur software version 2.0.7 (Thermo Scientific) was used to acquire mass spectrometry data.
Pinpoint software version 10 was used to extract areas under the curve. Mass spectra were analyzed using MaxQuant. Peptides and proteins were thrown out if they failed to meet the 1% false positive rate.
Candidate proteins were searched in UniProt, Human Protein Reference Database, and Entrez Gene databases. Results of the study revealed 904 proteins with differential expression. These proteins corresponded to 25 molecular pathways, which included development of cell morphology, immune response, tissue development, and development of the hematological system. In addition, proteins were identified with correlations to cancer, neurological disease, cardiovascular disease, metabolic disease, and genetic disorders.
Of these proteins, 60 showed atypical expression in Trisomy 21 amniocytes. This indicated a potential role in Down syndrome pathogenesis. In particular, SRM analysis revealed differential expression of two proteins: SOD1 and NES.
1. Underwood, M.A., et al. (2005) ‘Amniotic fluid: not just fetal urine anymore‘, Journal of Perinatology, 25 (5), (pp. 341–348)
2. Cho, C.K., et al. (2013) ‘Quantitative proteomic analysis of amniocytes reveals potentially dysregulated molecular networks in down syndrome‘, Clinical Proteomics, 10 (1), (p. 2)