Down sydrome (DS) is a relatively frequent chromosomal disorder. It is diagnosed in 1:500 to 1:800 of pregnant females (1). DS is clinically manifested by multiple somatic anomalies, mental retardation and precocious dementia. Pathological examination of DS brains reveals poor maturation, atrophy of the dendrites and the early appearance of senile plaques, which are characteristic of Alzheimer’s disease (AD). DS represents an important issue for affected families and the society, but there is no effective treatment at present. Advanced maternal age (35 years old) is an important risk factor for fetal DS (2). The most popular diagnostic strategy for DS relies in the combined detection of AFP, hCG, uE3 and inhibin A, with a detection rate of 60–75% and a 5% false positive rate (3). It is important to find a safe and effective method for diagnosis of DS. One strategy to achieve this is via identification of new biomarkers using noninvasive proteomic approaches. In a previous study, the isobaric tags for relative and absolute quantification (iTRAQ) technique was combined with matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF)/TOF select biomarkers (4). In this study, iTRAQ was combined with Gene Ontology (GO) analysis, in order to identify the proteins that are differentially expressed in DS and their predicted functions.

Proteomic approaches are a promising tool for the identification of diagnostic biomarkers of DS (5). iTRAQ was developed by Applied Biosystems, Inc. (Foster City, CA, USA) in 2004. iTRAQ-based proteomic analysis represents a major development in the rapid detection of potential biomarkers. The key advantage of the 8-plex iTRAQ system is the ability to simultaneously analyze up to 8 different biological specimens, thereby increasing the throughput while reducing experimental errors (6). iTRAQ has been recently employed in the study of certain diseases (7–9). In this study, we used iTRAQ in conjunction with multidimensional chromatography, followed by GO analysis, to detect and quantify proteome differences.

Quantification of the proteins via iTRAQ analysis has been suggested to be a suitable strategy for the identification of biomarkers, since iTRAQ allows the comparison of protein abundance among samples by measuring the peak intensities of reporter ions released from the iTRAQ-tagged peptides. In this study, we adopted iTRAQ technology and GO analysis to quantitatively analyse the proteome of plasma from the umbilical cord blood of DS fetus-carrying mothers, in order to identify useful biomarkers for DS. As a result, 505 proteins were identified, and were classified to MF, CC and BP terms by GO analysis. Among the up- and downregulated proteins in DS (Table I), we focused on 5 (apolipoprotein E, CFB, APCS, matrin-3, and OPN) to verify whether these may constitute novel DS biomarkers.

A putative risk factor for AD in the general population, the E4 allele of the apolipoprotein E gene, has highlighted the role of genetic influences in AD. It has long been recognized that DS is associated with early and severe development of AD neuropathological lesions (12). APCS and the Ig λ chain C region were also found in the sera of patients with DS in another study (8). We found that the concentration of apolipoprotein E in the plasma of umbilical cord blood was 3.75 times (x) higher in the DS compared to the control group The MF, CC and BP GO terms associated with apolipoprotein E are shown in Table III.

CFB plays an important role in the alternative complement pathway in AD (13), which shares a number of similar features with DS. Yu et al (14) found that the concentration of CFB is significantly increased in the serum of mothers with fetuses affected by DS. CFB may be associated with the brain damage that occurs in DS. In certain studies, CFB was associated with brain diseases, and a relationship between DS and the complement system was revealed (13,15–17). In the present study, we found that the concentration of CFB in the plasma of umbilical cord blood was 1.95× higher in the DS compared to the control group. The MFs associated with CFB were protein binding and hydrolase activity. The CCs associated with CFB were intracellular part, intracellular and protein complex. The BPs associated with CFB included activation of immune response, immune effector process, and response to stress (full list in Table III).

The APCS glycoprotein is encoded by a single gene locating on the human chromosome 1, has 204 residues, and is secreted (18). APCS immunoreactivity is commonly observed in DS, AD, and amyloid disorders with primarily cerebrovascular compromise (19,20). In the present study, we found that the concentration of APCS in the plasma of umbilical cord blood was 1.68× higher in the DS compared to the control group. The MFs, CCs and BPs associated with APCS are listed in Table III. The MFs were protein and carbohydrate binding, the CCs were protein complex, extracellular space and extracellular region part, and the BPs included macromolecular complex subunit organization, CC assembly, response to stress, and protein complex biogenesis (Table III).

OPN is a secreted glycoprotein with an arginine-glycine-aspartic acid (RGD) sequence, and able to bind a number of receptors, including the integrins and certain variant forms of CD44. OPN plays an important role in diverse fibro-inflammatory diseases. OPN is expressed in hepatocytes and macrophage-like cells in DS individuals showing perinatal liver fibrosis. OPN was thus proposed to be involved in the pathogenesis of perisinusoidal liver fibrosis, frequently observed in neonates with DS (21). We found that the concentration of OPN in the plasma of umbilical cord blood was 2.27× lower in the DS compared to the control group. The MFs, CCs and BPs associated with OPN are displayed in Table III. The MFs were protein and extracellular matrix binding, the CCs included intracellular part, intracellular, and extracellular space, while the BPs included response to stress, external stimulus, and chemical stimulus (Table III).

Matrin-3 is an inner nuclear matrix protein of 125 kDa, which is highly conserved in mammals (22). Matrin-3 contains a bipartite nuclear localization signal, two zinc finger domains, and two canonical RNA recognition motifs (23). Matrin-3 is encoded by the MATR3 gene, which is expressed in skeletal muscle (24). Autosomal and the active X chromosome territories were found to express matrin-3 (25). Matrin-3 was also found to be a target of the ataxia telangiectasia mutated (ATM) protein kinase (26). Degradation of matrin-3 may be a key cellular event, which induces a shift from apoptotic to necrotic death (27). Matrin-3 expression was significantly decreased in the fetal DS brain (28). The manifold decreased spot unambiguously assigned to matrin-3 in previous studies may reflect or induce aberrant transcription reported to occur in DS (28,29). In this study, we found that the concentration of matrin-3 in the plasma of umbilical cord blood was 100× lower in the DS compared to the control group. We did not found a BP term associated with matrin-3. The MFs associated with matrin-3 were protein and nucleotide binding, and the CCs included intracellular part, intracellular, and organelle part (Table III).

In conclusion, the iTRAQ technology, a relatively new strategy for proteomic analysis, was used here to study the changes in protein expression associated with DS. This approach identified 5 significantly differentially expressed proteins (apolipoprotein E, CFB, APCS, matrin-3 and OPN), which were previously reported to relate to DS. The MFs, CCs and BPs associated with these proteins indicate that they may be suitable DS biomarkers. However, further investigation of the functions of these proteins, as well as of the proteins with high fold changes in DS identified herein is required.