Proteomic Identification of Novel Differentiation Plasma Protein Markers in Hypobaric Hypoxia-Induced Rat Model

Abstract

BackgroundHypobaric 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.MethodsIn 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.ResultsBioinformatics 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/SignificanceThis 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.

Revisiting cobalt chloride preconditioning to prevent hypobaric hypoxia-induced damage: identification of global proteomic alteration and key networks

“…Briefly, the 500 ug of precipitated plasma protein from each sample was separated in first dimension using I m m o b i l i n e D r y S t r i p s ( 1 7 c m , p H 4 -7 , G E Healthcare, Sweden) strictly, as per the condition described previously by Ahmad et al (Ahmad and Sharma 2009;Ahmad et al 2014Ahmad et al , 2015, followed by second dimension separation on polyacrylamide gels.…”

“…After electrophoresis, proteins were visualized by modified silver staining procedure compatible with MS (Villanueva et al 2004) previously described by Ahmad et al (Ahmad and Sharma 2009;Ahmad et al 2014Ahmad et al , 2015. Briefly, the gels were fixed in a fixing solution (50 % v/v methanol, 12 % v/v acetic acid and 0.05 % v/v formaldehyde).…”

“…In order to perform analysis and calculate relative abundance of proteins, stained gels were imaged using an Investigator TM ProPic II Genomics Solutions robotic scanner. The 16-bit grey scale TIFF images were than processed with 2-D progenesis analysis software packages as described previously (Ahmad and Sharma 2009;Ahmad et al 2014). A modified method suggested by Turko et al (Turko and Murad 2003) was used for identification of significance (p ≤ 0.05, oneway ANOVA) in a consistent direction (increase or decrease).…”

“…The in-gel digestion was performed as described by Ahmad et al previously (Ahmad and Sharma 2009;Ahmad et al 2014). In brief, extracted spots from the gel were washed extensively with ion-free water.…”

“…The MS analysis was then performed as described previously (Ahmad and Sharma 2009;Ahmad et al 2014) by Ahmad et al Briefly, the dried peptides were resuspended in 5 ul of 0.1 % TFA and each spot of interest was mixed with an acidic α-cyano-4-hydroxy cinnamic acid (CHCA) matrix at final concentration of 10 mg/ml prepared in 70 % acetonitrile and 0.03 % TFA. Peptide mixture was then mixed with matrix solution in equal volume and loaded onto target plate.…”