| Objective:The aim of our study is to examine the hypothesis that plasma proteins may be altered and related to the pathologic changes of isolated ventricular septal defect (VSD). We will search for plasma differential proteins using the proteomic methods and analyze these proteins’ possible clinic implications in treatment and future diagnostic biomarker. Through this research, we discuss and summarize the research methods in proteomics technology in congenital heart disease, for application in the study of complex congenital heart disease in the future to lay the foundation.Methods:The study included55consecutive isolated VSD patients and55control healthy children with the same ethnicity and gender. Differential proteins analysis was performed in the plasma of patients with VSD and normal controls by using two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). Blood samples were taken from patients preoperatively. From each sample,2mL blood was allowed to clot at4℃for at least2hours and then centrifuged at3000rpm for5minutes to reach sediment of the clotted cells. Plasma was then collected, divided into aliquots, and stored frozen at-80℃until the analysis was done. Plasma samples from15isolated VSD patients and15healthy controls were separately pooled in each group and processed to deplete the top two (albumin, IgG) high-abundance proteins using the Albumin/IgG Removal Kit. Then2-DE was done3times in each pooling sample. After2-DE separation, the6gels were stained with Coomassie Blue R-250. Spot detection and quantification were done using PD-Quest2DE-analysis software. Protein spots were excised from the gel and were digested overnight in trypsin. The peptides were extracted and analyzed on a MALDI-TOF MS. These mass spectra were interpreted with GPS Explorer software for protein identification by peptide mass fingerprinting (PMF). Candidate differential proteins that might relate to disease processes were farther confirmed by enzyme-linked immunosorbent assays(ELISA)in erdarging samples(n=40).Results:Four differentially expressed proteins were identified by2-DE and MS, of which3downregulated proteins are Ficolin-3、Hp、SAP and1upregulated protein is α1-AGP. The plasma levels of SAP (3.818±0.2294ng/ml vs6.270±0.7723ng/ml; p=0.0029, n=40in each group), Hp (0.3965±0.04186mg/ml vs0.5994±0.07097mg/ml; p=0.016, n=40in each group), Ficolin-3(4.93±0.36mg/mL vs10.58±1.58mg/mL in control; p=0.001, n=40in each group) in VSD patients which were measured by ELISA were significantly lower than those in normal controls. At the same time the plasma levels of α1-AGP (3.086±0.1294mg/ml vs2.302±0.1071mg/ml in control; p<0.0001, n=40in each group) was significantly higher than that in normal control.Conclusions:We used proteomic methods to demonstrate the plasma protein changes in isolated VSD patients and be further confirmed by ELISA experiment. These differentially expressed proteins have strong clinical implications. The only upregulated protein α1-AGP may be associated with the VSD children relatively stress state. These downregulated proteins Ficolin-3%Hp、SAP may reveal the possible mechanisms for the susceptibility to pulmonary infections and pulmonary hypertension in patients. This study of differentially expressed proteins has practical significance for further understanding the pathological changes of VSD and points out the direction for the further research of the mechanism and biomarkers. Moreover, through our research proves that proteomics technologies have its application value in the study of CHD. |
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