Proteomic identification of novel differentiation plasma protein markers in hypobaric hypoxia-induced rat model

Abstract

Background: Hypobaric hypoxia causes complex changes in the expression of genes, including stress related genes and corresponding proteins that are necessary to maintain homeostasis. Whereas most prior studies focused on single proteins, newer methods allowing the simultaneous study of many proteins could lead to a better understanding of complex and dynamic changes that occur during the hypobaric hypoxia.

Methods: In this study we investigated the temporal plasma protein alterations of rat induced by hypobaric hypoxia at a simulated altitude of 7620 m (25,000 ft, 282 mm Hg) in a hypobaric chamber. Total plasma proteins collected at different time points (0, 6, 12 and 24 h), separated by two-dimensional electrophoresis (2-DE) and identified using matrix assisted laser desorption ionization time of flight (MALDI-TOF/TOF). Biological processes that were enriched in the plasma proteins during hypobaric hypoxia were identified using Gene Ontology (GO) analysis. According to their properties and obvious alterations during hypobaric hypoxia, changes of plasma concentrations of Ttr, Prdx-2, Gpx -3, Apo A-I, Hp, Apo-E, Fetub and Nme were selected to be validated by Western blot analysis.

Results: Bioinformatics analysis of 25 differentially expressed proteins showed that 23 had corresponding candidates in the database. The expression patterns of the eight selected proteins observed by Western blot were in agreement with 2-DE results, thus confirming the reliability of the proteomic analysis. Most of the proteins identified are related to cellular defense mechanisms involving anti-inflammatory and antioxidant activity. Their presence reflects the consequence of serial cascades initiated by hypobaric hypoxia.

Conclusion/significance: This study provides information about the plasma proteome changes induced in response to hypobaric hypoxia and thus identification of the candidate proteins which can act as novel biomarkers.

Figure 1. A representative 2D gel of plasma proteins from hypobaric hypoxia treated rat, with a pH range from 5–8.

Distribution of differentially expressed protein spots and each spot number relates to data shown in Table 1.

Figure 2. Magnified comparison maps of (A) spot 24, (B) spot 8 and (C) spot 17 in the 2-DE patterns with samples obtained at different time points after exposure to acute hypobaric hypoxia.

Spot 24 was expressed in the control total plasma proteins but disappeared after acute hypobaric hypoxia. Spot 8 had low expression in the control group but its expression increased at each time point after acute hypobaric hypoxia. Spot 17 had high expression in the control group, but its expression decreased steadily after acute hypobaric hypoxia.

Figure 3. Gene ontology annotations of the proteins identified by MALDI-TOF/MS.

Results were obtained using Blast2GO annotation. The distributions of identified proteins according to their (A) molecular functions and (B) biological processes are shown.

Figure 4. Validation of protein change patterns at different time points by Western blot analysis of proteins Ttr, Prdx-2, Gpx-3, Apo A -1, Hp, Apo-E, Fetub and Nme.

Total plasma proteins (40 µg/lane) were separated by SDS-PAGE and probed with primary antibodies against these eight proteins.