Comparative proteomic analysis of human amniotic fluid supernatants with Down syndrome using mass spectrometry.

Department of Applied Biochemistry, Konkuk University, Chungju 380-701, Korea Received: December 28, 2009 / Revised: February 12, 2010 / Accepted: February 15, 2010Down syndrome (DS) is an abnormality of the 21stchromosome that commonly occurs in children born toolder women. Thus, amniotic fluid (AF) is usually collectedfrom such women for prenatal diagnosis. This studyanalyzed human AF supernatants (AFS) using a massspectrometric (MS) approach to search for candidatebiomarkers of a DS pregnancy. The AFS were collectedfrom older pregnant women at weeks 16-18 of theirgestation by amniocentesis for cytogenetic analysis. The AFSfrom the pregnancies carrying DS (n=4) or chromosomallynormal (n=6) fetuses, as revealed by the cytogenetic analysis,were then subjected to global protein profiling based onliquid chromatography-electrospray ionization-tandem massspectrometry (LC-ESI-MS/MS). Affinity chromatographywas also applied prior to the LC -ESI-MS/MS to minimizethe masking effect of highly abundant albumin andimmunoglobulin and thereby increase the diversity of theidentified proteins. As a result, at least 30 new AFS proteinswere identified and 44 AFS proteins were found to bedifferentially expressed between the DS and normal cases,where 6 of the proteins were unique to the DS cases and 11were unique to the chromosomally normal cases. In addition,in the DS cases, 19 AFS proteins were downregulated and8 were upregulated to varying degrees. A Western blotanalysis confirmed the LC-ESI-MS/MS data, indicatingthat the combined detection of apolipoprotein A-II (apoA-II) and alpha-fetoprotein (AFP) could be a potentialtool for diagnosing DS cases. Keywords: Proteomics, biomarker, Down syndrome, amnioticfluid, liquid chromatography-electrospray ionization-massspectrometry.Down syndrome (DS) is an abnormality of the 21st chromosomein humans and is characterized by impaired cognitiveability, physical growth, facial appearance, and gonadalfunction in both male and females [5, 16, 32, 36, 39]. Reportedto occur in about 1 in 800 live births, the incidence increaseswith an increase in the maternal age [15, 17, 52]. DS offspringare generally infertile or subfertile, although there are afew reports of live births from DS men [32, 39]. DS is usually diagnosed by screening the maternal serumfor alpha-fetoprotein (AFP), human chorionic gonadotropin(hCG), and dimeric inhibin A, with ~22-25% false-negativeand ~7.5-8.5% false-positive rates [4]. In families with anincreased chance of having a DS child, more invasivetechniques, such as amniocentesis, chorionic villus sampling,or percutaneous umbilical cord blood sampling, are employedfor the diagnosis. Analysis of the amniotic fluid (AF),obtained by amniocentesis, also facilitates prenatal geneticdiagnosis and can reveal inflammatory conditions affectingthe pregnancy. Moreover, proteomic profiling of the AF hasbeen shown to be useful in detecting inflammation, infection,and neonatal sepsis [6, 7], along with the identification ofspecific biomarkers and diagnostic profiling of pathophysiologicconditions of the pregnancy, such as Rh(