RESEARCH ARTICLE Proteomic analysis of amniotic fluid in pregnancies with Down syndrome George Th. Tsangaris 1 , Panagiotis Karamessinis 1 , Aggeliki Kolialexi 2 , Spiros D. Garbis 1 , Aris Antsaklis 3 , Ariadni Mavrou 2 and Michael Fountoulakis 1 1 Division of Biotechnology, Centre of Basic Research, Foundation for Biomedical Research of the Academy of Athens, Athens, Greece 2 Medical Genetics, Athens University School of Medicine, Athens, Greece 3 First Department of Obstetrics and Gynecology, Athens University School of Medicine, Athens, Greece Proteomic analysis is widely used for the detection of diagnostic markers. In the present study amniotic fluid supernatants (AFS) from pregnancies with Down syndrome (DS) fetuses and from chromosomally normal fetuses in the 17th week of gestation were analyzed by 2-DE. Gel compar- ison revealed significant differences in the two groups. Spots with different expression levels were excised and proteins were identified by MALDI-MS and nano-ESI-MS/MS. Splicing factor arginine/serine-rich 4 (SFRS4; Q08170) was present only in AFS from DS fetuses and completely absent in the control group. Quantitative differences were detected for alpha-1-microglobulin (AMBP; P02760), collagen alpha 1 (I) chain (CO1A1; P02452), collagen alpha 1 (III) chain (CO3A1; P02461), collagen alpha 1 (V) chain d (CO5A1; P20908), and basement membrane- specific heparin sulfate proteoglycan core protein (PGBM; P98160). These proteins were increased in cases with DS, whereas protein IBP-1 (P08833) was decreased by 40% compared with chromo- somally normal fetuses. Four proteins, CO1A1, CO3A1, CO5A1, and PGBM, appeared as frag- ments. As differentially expressed proteins were present in all pregnancies with DS tested, they may represent useful potential markers for prenatal diagnosis. However, for protein biomarkers to be of any clinical utility, systematic analysis of the maternal serum should be conducted. Received: February 1, 2006 Revised: April 7, 2006 Accepted: May 11, 2006 Keywords: Amniotic fluid / Biomarkers / Down syndrome / Prenatal diagnosis 4410 Proteomics 2006, 6, 4410–4419 1 Introduction In the postgenomic era, medical research has moved beyond the one-gene-at-a-time analysis and is currently profiling thou- sands of gene products to give a global perspective of a particu- lar clinical condition. MS-based proteomic analysis represents a major development in the rapid detection of potential bio- markers. Proteomic diagnostics combine pattern profiling of tissue and body fluids by MS with sophisticated bioinformatic tools in order to identify patterns within the complex proteomic profile that can discriminate between normal, benign, and dis- ease states. Additionally, MS-based proteomics stand to Correspondence: Dr. George Th. Tsangaris, Division of Biotech- nology, Foundation for Biomedical Research of the Academy of Athens, Soranou Efesiou 4, 115 27 Athens, Greece E-mail: gthtsangaris@biocademy.gr Fax: 130-210-6597546 Abbreviations: AFS, amniotic fluid supernatant; AMBP , alpha-1- microglobulin; CO1A1, collagen alpha 1 (I) chain; CO3A1, col- lagen alpha 1 (III) chain; CO5A1, collagen alpha 1 (V) chain d; DS, Down syndrome; IBP-1, insulin-like growth factor binding protein 1; PGBM, basement membrane-specific heparin sulfate proteoglycan core protein; SFRS4, splicing factor arginine/se- rine-rich 4 DOI 10.1002/pmic.200600085  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com