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

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Proteomic Identification of Novel Differentiation Plasma Protein Markers in Hypobaric Hypoxia-induced Rat Model
  Proteomic Identification of Novel Differentiation PlasmaProtein Markers in Hypobaric Hypoxia-Induced RatModel Yasmin Ahmad 1 * . , Narendra K. Sharma 1 . , Mohammad Faiz Ahmad 2 , Manish Sharma 1 , Iti Garg 3 ,Kalpana Bhargava 1 1 Peptide and Proteomics Division, DIPAS, DRDO, Ministry of Defence, Delhi, India,  2 Department of Chemistry, Jamia Millia Islamia, New Delhi, India,  3 Department of Genomics, DIPAS, DRDO, Ministry of Defence, Delhi, India Abstract Background:   Hypobaric hypoxia causes complex changes in the expression of genes, including stress related genes andcorresponding 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 anddynamic 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 asimulated altitude of 7620 m (25,000 ft, 282 mm Hg) in a hypobaric chamber. Total plasma proteins collected at differenttime points (0, 6, 12 and 24 h), separated by two-dimensional electrophoresis (2-DE) and identified using matrix assistedlaser desorption ionization time of flight (MALDI-TOF/TOF). Biological processes that were enriched in the plasma proteinsduring hypobaric hypoxia were identified using Gene Ontology (GO) analysis. According to their properties and obviousalterations during hypobaric hypoxia, changes of plasma concentrations of Ttr, Prdx-2, Gpx -3, Apo A-I, Hp, Apo-E, Fetub andNme were selected to be validated by Western blot analysis. Results:   Bioinformatics analysis of 25 differentially expressed proteins showed that 23 had corresponding candidates in thedatabase. The expression patterns of the eight selected proteins observed by Western blot were in agreement with 2-DEresults, thus confirming the reliability of the proteomic analysis. Most of the proteins identified are related to cellulardefense mechanisms involving anti-inflammatory and antioxidant activity. Their presence reflects the consequence of serialcascades initiated by hypobaric hypoxia. Conclusion/Significance:   This study provides information about the plasma proteome changes induced in response tohypobaric hypoxia and thus identification of the candidate proteins which can act as novel biomarkers. Citation:  Ahmad Y, Sharma NK, Ahmad MF, Sharma M, Garg I, et al. (2014) Proteomic Identification of Novel Differentiation Plasma Protein Markers in HypobaricHypoxia-Induced Rat Model. PLoS ONE 9(5): e98027. doi:10.1371/journal.pone.0098027 Editor:  Jo¨rn Karhausen, Duke University Medical Center, United States of America Received  December 23, 2013;  Accepted  April 28, 2014;  Published  May 19, 2014 Copyright:    2014 Ahmad et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the srcinal author and source are credited. Funding:  Financial support for this study is provided by a grant from TC/321/Task –145 (YA)/DIPAS/2008, Defence Research Development Organization (DRDO),Ministry of Defence, Government of India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of themanuscript. Competing Interests:  The authors have declared that no competing interests exist.* E-mail: .  These authors contributed equally to this work. Introduction High altitude is characterized as a region of low barometricpressure (hypobaric), low partial pressure of oxygen (hypoxia),severe cold and increase in ultraviolet radiation. High altitudeposes several operational problems to the sojourners, soldiers andmountaineers not only during their initial days of induction to thehypoxic environment but also followed prolonged residency. Withan increase in altitude, atmospheric pressure and the partialpressure of oxygen decrease rapidly leading to decreased O 2 availability. This thus results in a condition termed as hypobarichypoxia which stresses biological systems because of non-availability of steady uninterrupted supply of oxygen formitochondrial metabolism. The cellular responses to hypobarichypoxia are complex and characterized by alteration in theexpression of a number of genes, including stress related genes andcorresponding proteins that are necessary to maintain homeostasis[1]. Genes and their products (mRNA and proteins) that respondto hypobaric hypoxia have a great potential to serve as indicatorsof hypoxic stress, including enzymes of the glycolytic pathway,(which increase anaerobic ATP production), glucose transporters,enzymes involved in amino acid metabolism and gluconeogenesis,(which maintain blood glucose levels) [2], and heat shock proteins(which are involved in protein stability and folding) [3]. In general,genes that encode proteins involved in energy production, proteinsynthesis and degradation, lipid and carbohydrate metabolism,locomotion and contraction, and antioxidant defense are also the PLOS ONE | 1 May 2014 | Volume 9 | Issue 5 | e98027  potential biomarkers of hypoxic stress [2]. Transcript levels of genes encoding specific proteins which can deal with perceivedstressors are usually the first measurable biomarkers that can beassessed. However, examining gene expression alterations by itself does not give a complete picture as it is also essential to quantifythe protein activity to ascertain that altered gene expression alsoresults in altered protein levels. Changes in specific geneexpression levels as well as the protein levels are excellentindicators that the organism has mobilized metabolic pathwaysin response to a specific stimulus. A broader understanding of hypoxia-induced alterations incellular or organ function could be better achieved from acombined knowledge derived from the concerted application of genomic and proteomics approaches. Although genomic changesduring hypoxia have been extensively investigated, hypoxia-induced changes in the proteome of mammalian cells are onlyin the early phase of investigation. So far, a large number of studieshave focused on the influence of hypoxia on the expression andposttranslational modification of a single protein of interest or asubset of functionally related proteins; however, very few reportshave really examined proteome-wide alteration during hypoxia,with most focussing on cell lines [4–6].The aim of the present study was to explore changes in theplasma proteome of rat exposed to hypobaric hypoxia at differenttime points (0 h, 6 h, 12 h and 24 hrs) and the levels of a specificprotein of interest following hypobaric hypoxia have beenmeasured by using proteomics tools. Plasma has the advantageover cells or tissue because it can be collected in a relatively non-invasive manner and has an immense diagnostic potential [7].Moreover, all the protein components are readily accessible in asingle compartment without requiring additional extractionprocedures. Additionally, its constituents reflect closely thephysiological and pathological alterations due to any stresscondition [8]. Advances in proteomic techniques have facilitatedthe investigation of global changes in plasma proteomes. A varietyof complementary procedures for the global analysis of proteinexpression have been described. These include two-dimensionalgel electrophoresis (2-DE) coupled with mass spectrometry (MS)[9], multidimensional chromatography coupled with tandem MS[10], and chip technologies coupled with either antigens [11] orantibodies [12]. Among them, 2-DE coupled with matrix-assistedlaser desorption/ionization MS is a prominent method for theidentification of the proteins as well as for quantification of changes in protein expression [9,13]. This proteomics analysis canprovide information pertaining to compensatory changes occur-ring at the level of protein expression by altering antioxidant/oxidant status as a consequence of prior transcriptional andtranslational alterations in response to hypoxia and environmentalperturbations. Results The protein expression profile in the plasma of hypoxia treatedand control rats were obtained by two-dimensional electrophoresis(2-DE) with linear IPG strips of 17 cm, and pH range from 5–8.Proteins were resolved according to their isoelectric point (pI) inthe first dimension and their molecular weight (Mw) in the seconddimension. Gel images and representative 2-DE maps wereunambiguously matched by the progenesis 2D-software, anddisplayed well-resolved and reproducible profiles for hypoxiatreated and control rats. Approximately 500–800 protein spotswere detected by silver staining in a single 2-DE gel and weredistributed across the pI range 5–8, with molecular massesbetween 10000 and . 130000 Da (Figure 1). On a 2-DE gel, oneprotein may be represented as a single spot or may be present asmultiple spots, due to changes in molecular mass caused by posttranslational modification of the primary protein product or as aresult of protein processing. Such modifications may also result inalterations of pI or conformation of a protein and consequentlycause a shift in its position on the 2-D gel, thus resulting in changeof spot intensity. Spots can also be considered in terms of spotfamilies, representing the multiple spots within a 2-DE proteinpattern created from a single primary protein. The quantity of each spot in a gel was normalized as a percentage of the totalquantity of all the spots in the gel. In comparison with 2-DEpatterns, protein spots with significant changes (p , 0.05, one–way ANOVA) in a consistent direction (increase or decrease) were judged as deregulated and were chosen for identification. Thesedifferentially expressed plasma proteins had three types of expression patterns: (1) expressed in the control but not detectedafter exposure to acute hypoxia, (2) expressed slowly in the controlgroup, but increased after exposure to acute hypoxia, irrespectiveof the time of exposure, and, (3) expressed highly in the controlgroup, but decreased steadily after acute hypoxia. Figure 2 shows amagnified comparison of the patterns of spot 24 (Figure 2A), spot 8(Figure 2B), and spot 17 (Figure 2C) which exemplify the maintypes of time-course for concentration levels after hypoxia. 25differential protein spots were detected, of which 16 spots were up-regulated (3, 4, 5, 8, 9, 10, 13, 14, 15, 16, 19, 21, 22, 23, 26 and27), while 9 spots were down-regulated (11, 12, 17, 18, 20, 24, 25,28 and 29). In the present study, we considered a $ 20% change assignificant and sufficient to include all the proteins for which evena 20% change in their expression levels (even at low fold change) islikely to have a functional relevance. Out of the 25 significantlyderegulated spots, 23 were successfully identified by MALDI-TOF/TOF with PMF and MS/MS analysis followed by databasesearching (Figure 1, Table 1). Table 1 summarizes the identifica-tion information for these identified protein spots including proteinname, accession number, molecular weight, pI value, and proteinfunction. For spots 25 and 27, corresponding proteins in thedatabase could not be found even after using the PMF and MS/MS searching. These may be novel proteins or else they may besmall fragments of some proteins as can be said based on theirmolecular weight (  $ 15 kDa). If they would have been only smallerfragments, the PMF information for these two spots could belimited resulting in the failure of detection of the corresponding proteins in the database. The differentially expressed proteinslisted here represent a wide range of biological categories. Proteinsrelated to cellular defense mechanisms involving anti-inflamma-tory and antioxidant activity were the most common. Whenorganized according to their molecular functions, 22% of theidentified proteins correspond to those involved in binding andenzymatic regulatory activity, 11% involved in antioxidant activityand 19% in transporter activity (Figure 3). We also categorizedproteins according to their biological processes; most abundantgroups of proteins correspond to those involved in homeostaticprocess (16%), cellular response to oxidative stress (6%), cellularresponse to reactive oxygen species (8%) and lipid metabolicprocess (14%). Regarding their intensity rates and cellularfunctions in the plasma of hypoxia treated rats, transthyretin(Ttr), peroxiredoxin-2 (Prdx-2), glutathione peroxidase-3 (Gpx-3), Apolipoprotein A-I (ApoA-1), haptoglobin (Hp), Apolipoprotein-E(Apo-E), fetuin–B (Fetub) and Nucleoside diphosphate kinase B(Nme) were validated by Western blotting. Proteomic Analysis Focus on Hypobaric HypoxiaPLOS ONE | 2 May 2014 | Volume 9 | Issue 5 | e98027  Validation of Differentially Expressed Proteins by WesternBlot Analysis We selected eight proteins Ttr, Prdx-2, Gpx-3, ApoA-I, Hp, Apo-E, Fetub and Nme, and further verified whether theexpression patterns of proteins observed in 2-DE gels paralleledthose validated by Western blot analysis. Band intensity wasmeasured using Quantity One 1-D Analysis Software version 4.6.7BIO-RAD, and the intensity ratio corresponding to  b  –tubulinband was calculated. Both Apo-E and Fetub were down-regulatedin plasma after exposure to acute hypobaric hypoxia (Figure 4).The protein levels of Fetub increased gradually from 6 to 12 hrbut down-regulated at 24 hr after acute hypobaric hypoxia. Theexpression of ApoA-I, Hp, Nme and Ttr increased gradually from6 to 24 hr after acute hypoxia (Figure 4). Both Prxd-2 and Gpx-3were up-regulated in plasma after acute hypobaric hypoxia andremained at high levels (Figure 4). The expression patterns of theselected proteins observed by Western blot were in agreement with2-DE results, thus confirming the reliability of the proteomicanalysis. Discussion High altitude exposure depends not only on the speed of ascent,degree of hypoxia but also on the duration of stay at a givenaltitude. It is therefore, important to decipher the temporalproteomic changes in plasma for a better understanding of severalaltitude-induced pathophysiological mechanisms in order to 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.doi:10.1371/journal.pone.0098027.g001 Figure 2. Magnified comparison maps of (A) spot 24, (B) spot 8and (C) spot 17 in the 2-DE patterns with samples obtained atdifferent time points after exposure to acute hypobarichypoxia.  Spot 24 was expressed in the control total plasma proteinsbut disappeared after acute hypobaric hypoxia. Spot 8 had lowexpression in the control group but its expression increased at eachtime point after acute hypobaric hypoxia. Spot 17 had high expressionin the control group, but its expression decreased steadily after acutehypobaric hypoxia.doi:10.1371/journal.pone.0098027.g002Proteomic Analysis Focus on Hypobaric HypoxiaPLOS ONE | 3 May 2014 | Volume 9 | Issue 5 | e98027  identify new biomarkers or potential therapeutic targets. To ourknowledge, this is the first comprehensive proteome studyreporting the proteome level changes in plasma of rats exposedto acute hypobaric hypoxia. Our results obtained by 2-DEelectrophoresis and partly also confirmed by the use of othertechniques, indicate that short-term acute hypobaric hypoxia notonly identify hypoxia-modulated early proteins but also altereddistinct biological process depending on the stress duration. Therelationships of these proteins with hypobaric hypoxia areelucidated in the following section. Regulation of Apolipoproteins in Plasma after HypobaricHypoxia We found several apolipoproteins to be regulated in plasma of rat exposed to hypobaric hypoxia. These proteins functionprimarily as lipid binding proteins to transport lipids from theintestine to the liver and from the liver to tissues, including adipocytes, lung, heart, muscle, and breast tissues. In recentstudies, apolipoproteins have been shown to have a clinicalimportance because of their roles in endothelial protection, anti-oxidation, and anti-inflammation [14,15]. Here, we evidence thedifferential expression of apolipoproteins A-I, M, E, A-IV and H atdifferent time points in plasma after acute hypobaric hypoxia.Specifically, apolipoprotein A-I (spot no.10) belong to the ApoA-I/ A4/E protein family and is primarily produced in the liver and theintestine. ApoA-I can be found in the extracellular space and,being a structural component of high density lipoprotein (HDL),takes part in cholesterol absorption. ApoA-I up-regulation isassociated with breast and lung cancer [16,17]. Interestingly,studies provide new evidence supporting the notion that HDLplays a protective role in the lung. ABCA1, which interacts withlipid-poor ApoA-I, was earlier shown to be essential formaintaining normal lipid composition and architecture of thelung as well as respiratory physiology [18]. There is emerging evidence for the critical role of ApoA-I in protecting pulmonaryartery and airway function as well as in preventing inflammationand collagen deposition in the lung [19]. More recently, proteomicstudies revealed the anti-inflammatory role of ApoA-I in HAPEpatients [20]. Here, we report ApoA-I expression increasedgradually from 6 to 24 hr after acute hypoxia suggesting theanti-inflammatory role of ApoA-I. Spot 11 was identified asapolipoprotein M (Apo-M) which is produced in the liver andsecreted in plasma. Apo-M is a member of the lipocalin family, agroup of proteins with a characteristic coffee filter-like structure Figure 3. Gene ontology annotations of the proteins identified by MALDI-TOF/MS.  Results were obtained using Blast2GO annotation. Thedistributions of identified proteins according to their (A) molecular functions and (B) biological processes are shown.doi:10.1371/journal.pone.0098027.g003Proteomic Analysis Focus on Hypobaric HypoxiaPLOS ONE | 4 May 2014 | Volume 9 | Issue 5 | e98027      T   a    b    l   e    1 .      L     i    s    t    o     f     d     i     f     f    e    r    e    n    t     i    a     l     l    y    e    x    p    r    e    s    s    e     d    r    a    t    p     l    a    s    m    a    p    r    o    t    e     i    n    s    a     f    t    e    r    t    r    e    a    t    e     d    w     i    t     h     h    y    p    o     b    a    r     i    c     h    y    p    o    x     i    a ,     i     d    e    n    t     i     f     i    e     d     b    y     M     A     L     D     I  -     M     S     /     M     S . 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     N    e    g    a    t     i    v    e    r    e    g    u     l    a    t     i    o    n    o     f     i    n     f     l    a    m    m    a    t    o    r    y    r    e    s    p    o    n    s    e ,    e    x     h     i     b     i    t    a    n    t     i    o    x     i     d    a    n    t    p    r    o    p    e    r    t    y     1     8     P     0     2     6     5     1     2     5     5     A    p    o     l     i    p    o    p    r    o    t    e     i    n     A  -     I     V     5 .     1     2     /     4     4     5 .     2     5     /     4     3     0 .     7     1       b      A    c    t     i    v    a    t     i    o    n    o     f     L     C     A     T     1     9     P     0     4     2     7     6     5     6     8     V     i    t    a    m     i    n     D  -     b     i    n     d     i    n    g    p    r    o    t    e     i    n     5 .     6     5     /     5     5     5 .     7     5     /     5     5     2 .     1     1     a      2 .     1     3     a      1 .     3     3       b      I    n    p     l    a    s    m    a ,     i    t    c    a    r    r     i    e    s    t     h    e    v     i    t    a    m     i    n     D    s    t    e    r    o     l    s    a    n     d    p    r    e    v    e    n    t    s    p    o     l    y    m    e    r     i    z    a    t     i    o    n    o     f    a    c    t     i    n     b    y     b     i    n     d     i    n    g     i    t    s    m    o    n    o    m    e    r    s     2     0     Q     9     Q     X     7     9     4     0     0     F    e    t    u     i    n  -     B     6 .     7     1     /     4     2     5 .     9     1     /     6     0     1 .     2     1     c      0 .     6     6       b      A    c    u    t    e    p     h    a    s    e    a    n    t     i  -     i    n     f     l    a    m    m    a    t    o    r    y    m    e     d     i    a    t    o    r     2     1     P     2     6     6     4     4     1     6     4     B    e    t    a  -     2  -    g     l    y    c    o    p    r    o    t    e     i    n     1     8 .     5     9     /     3     4     6 .     9     8     /     5     7     0 .     5     6       b      P     l    a    s    m     i    n    o    g    e    n    a    c    t     i    v    a    t     i    o    n ,     2     2     Q     4     9     8     E     0     1     2     7     T     h     i    o    r    e     d    o    x     i    n     d    o    m    a     i    n    c    o    n    t    a     i    n     i    n    g    p    r    o    t    e     i    n     1     2     5 .     2     5     /     1     9     7 .     4     9     /     3     3     1 .     4     6       b      1 .     4     3       b      1 .     2     4     c      C    e     l     l    r    e     d    o    x     h    o    m    e    o    s    t    a    s     i    s     2     3     P     1     7     9     8     8     1     5     0     S    u     l     f    o    t    r    a    n    s     f    e    r    a    s    e     1     A     1     6 .     3     7     /     3     4     7 .     2     7     /     3     2     1 .     6     0     a      1 .     3     3       b      R    e    s    p    o    n    s    e    t    o    s    t    r    e    s    s ,    r    e    g    u     l    a    t     i    o    n    o     f     b     l    o    o     d    p    r    e    s    s    u    r    e     2     4     P     0     8     6     4     9     1     0     1     C    o    m    p     l    e    m    e    n    t     C     4     6 .     9     9     /     1     9     3     7 .     3     /     3     0     0 .     5     5       b      0 .     5     3     a      I    n     f     l    a    m    m    a    t    o    r    y    r    e    s    p    o    n    s    e ,    c    o    m    p     l    e    m    e    n    t    a    c    t     i    v    a    t     i    o    n     2     6     P     1     9     8     0     4     2     4     4     N    u    c     l    e    o    s     i     d    e     d     i    p     h    o    s    p     h    a    t    e     k     i    n    a    s    e     B     6 .     9     2     /     1     7     7 .     0     5     /     1     6     1 .     6     1     a      1 .     8     9     a      1 .     3     5       b      C    e     l     l    u     l    a    r    r    e    s    p    o    n    s    e    t    o    o    x     i     d    a    t     i    v    e    s    t    r    e    s    s ,    n    e    g    a    t     i    v    e    r    e    g    u     l    a    t     i    o    n    o     f    a    p    o    p    t    o    t     i    c    p    r    o    c    e    s    s     2     8     P     2     0     7     6     7     1     8     8     I    g     l    a    m     b     d    a  -     2    c     h    a     i    n     C     5 .     7     6     /     1     1     6 .     6     2     /     2     8     0 .     6     6       b      A    n    t     i    g    e    n     b     i    n     d     i    n    g     2     9     P     2     0     7     6     7     1     8     8     I    g     l    a    m     b     d    a  -     2    c     h    a     i    n     C     5 .     7     6     /     1     1     6 .     5     2     /     2     9     0 .     7     6     c      A    n    t     i    g    e    n     b     i    n     d     i    n    g      a      d    e    n    o    t    e    s     P      ,      0 .     0     0     1 ,        b      d    e    m    o    t    e    s    p      ,      0 .     0     1    a    n     d      c      d    e    n    o    t    e    s    p      ,      0 .     0     5 .     d    o     i   :     1     0 .     1     3     7     1     /     j    o    u    r    n    a     l .    p    o    n    e .     0     0     9     8     0     2     7 .    t     0     0     1 Proteomic Analysis Focus on Hypobaric HypoxiaPLOS ONE | 5 May 2014 | Volume 9 | Issue 5 | e98027
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