Comparative Proteomic Assessment of Normal vs. Polyhydramnios Amniotic Fluid Based on Computational Analysis

Abstract

Mass spectrometry-based proteomics have become a valued tool for conducting comprehensive analyses in amniotic fluid samples with pathologies. Our research interest is the finding and characterization of proteins related to normal vs. polyhydramnios (non-immune hydrops) pregnancy. Proteomic analysis was performed on proteins isolated from fresh amniotic fluid samples. Proteins were fractionated by 2DE using a different pI range (pI 3-11, pI 4-7) and analyzed with MALDI-TOF-MS. Furthermore, by using computational analysis, identified proteins in protein maps specific to normal vs. polyhydramnios pregnancy were compared and the quantities of expressed proteins were evaluated mathematically. Comparative analysis of proteome characteristic for the same polyhydramnios pregnancy fractionated by 2DE in different pI range (3-11 and 4-7) was performed and particular protein groups were evaluated for the quantification of changes within the same protein level. Proteins of normal and polyhydramnios pregnancies were fractionated by 2DE in pI range 3-11 and in pI range 4-7. Mass spectrometry analysis of proteins has revealed that the quantity changes of the main identified proteins in normal vs. polyhydramnios pregnancy could be assigned to immune response and inflammation proteins, cellular signaling and regulation proteins, metabolic proteins, etc. Specifically, we have identified and characterized proteins associated with heart function and circulatory system and proteins associated with abnormalities in prenatal medicine. The following are: serotransferrin, prothrombin, haptoglobin, transthyretin, alpha-1-antitrypsin, zinc-alpha-2-glycprotein, haptoglobin kininogen-1, hemopexin, clusterin, lumican, afamin, gelsolin. By using computational analysis, we demonstrated that some of these proteins increased a few times in pathological pregnancy. Computer assistance analysis of 2DE images suggested that, for the better isolation of the proteins' isoforms, those levels increased/decreased in normal vs. polyhydramnios pregnancy, and the fractionation of proteins in pI rage 3-11 and 4-7 could be substantial. We analyzed and identified by MS proteins specific for normal and polyhydramnios pregnancies. Identified protein levels increased and/or modification changed in case of non-immune hydrops fetus and in cases of cardiovascular, anemia, growth restriction, and metabolic disorders. Computational analysis for proteomic characterization empower to estimate the quantitative changes of proteins specific for normal vs. polyhydramnios pregnancies.

Keywords: amniotic fluid; computational analysis; mass spectrometry; polyhydramnios.

Figure 1

Comparative analysis of 2DE proteome maps characteristic for normal and polyhydramnios pregnancies. The proteins were resolved by 2DE, pH range 3–11, and Excel Gel SDS, gradient 8–18%. 2DE images of proteins of amniotic fluid of normal pregnancy (AFN, G1; blue in G1 + G2 and amniotic fluid of polyhydramnios pregnancy (AFP, G2; orange in G1 + G2) were superposed and presented in G1 + G2. Arrows and numbers in the 2DE maps indicate the positions of proteins supplied to MALDI-TOF MS/MS and identified. Spot labels are the same as in Table 1. Molecular weight (Mw) markers are presented on the left. Representative images from one of three experiments showing similar results are shown.

Figure 2

Comparative analysis of 2DE protein maps corresponding amniotic fluid of polyhydramnios pregnancy fractionated in different pI range. (A) proteins corresponding amniotic fluid of polyhydramnios pregnancy (AFP), fractionated in different pI range: pI 3–11 (G2, blue in G2 + G3) and pI 4–7 (G3, orange in G2 + G3) range and Excel Gel SDS, gradient 8–18%. Arrows and numbers in the 2DE maps indicate the positions of proteins supplied to MALDI-TOF MS/MS and identified. Spot labels for the proteins fractionated in the range pI 3–11 are the same as in Table 2. Spot labels for the proteins fractionated in the range pI 4–7 are the same as in Table 2. (B) Computational analysis of several protein groups is performed to evaluate their distribution in different pI value ranges. It shows that the same protein level with different pI changes because of modification level. In Table 2, the proteins’ spot distribution corresponding to the different modification level is presented (column— Share, %). Representative images from one of three experiments showing similar results are shown.

Figure 3

Functions of proteins identified in normal vs. polyhydramnios pregnancies, proteins fractionated in the pI 3–11 range. Proteins were clustered according to their functions by using AgBase [2.00 v].

Figure 4

Functions of proteins identified in normal vs. polyhydramnios pregnancies, proteins fractionated in the pI 4–7 range. Proteins were clustered according to their functions by using AgBase [2.00 v].