Research Article Comparative Proteomic Study Reveals the ...

Research ArticleComparative Proteomic Study Reveals the Molecular Aspects ofDelayed Ocular Symptoms Induced by Sulfur Mustard

Zaiddodine Pashandi1 Neda Saraygord-Afshari1

Hossein Naderi-Manesh1 and Mostafa Naderi2

1Department of Biophysics Faculty of Biology Tarbiat Modares University PO Box 14115-175 Tehran Iran2Department of Ophthalmology Chemical Research Center Baqiyatallah University of Medical Sciences Tehran Iran

Correspondence should be addressed to Hossein Naderi-Manesh nadermanmodaresacir

Received 14 July 2014 Accepted 10 December 2014

Academic Editor Christian Huck

Copyright copy 2015 Zaiddodine Pashandi et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Objective Sulfur mustard (SM) is a highly reactive alkylating agent which produces ocular respiratory and skin damages Eyesare the most sensitive organ to SM due to high intrinsic metabolic and rapid turnover rate of corneal epithelium and aqueous-mucous interfaces of the cornea and conjunctiva Here we investigate underlying molecular mechanism of SM exposure delayedeffects which is still a controversial issue after about 30 yearsMaterials and Methods Following ethical approval we have analyzedserumproteome of ten severe SM exposedmale patients with delayed eye symptomswith two-dimensional electrophoresis followedby matrix-assisted laser desorptionionization time-of-flighttime-of-flight mass spectrometry The western blotting was used toconfirm the proteins that have been identified Results We have identified thirteen proteins including albumin haptoglobin andkeratin isoforms as well as immunoglobulin kappa chain which showed upregulation while transferrin and alpha 1 antitrypsinrevealed downregulation in these patients in comparisonwith healthy control groupConclusionsOur results elevated participationof free iron circulatory imbalance and local matrix-metalloproteinase activity in development of delayed ocular symptoms inducedby SM It demonstrates that SM induced systemic toxicity leads to some serum protein changes that continually and graduallyexacerbate the ocular surface injuries

1 Introduction

Sulfur mustard (221015840-dichloroethylsulfide SM) is a bifunc-tional alkylating and vesicant agent with the highest militarysignificance After the First World War Iraqrsquos military usageof SM against Iranian military and civilians as well as theirown Kurdish civilians (1980ndash1988) was the most extensiveusage of this agent The outcome of this genocide was over100000 chemical warfare victims that have been groupedinto three categories of injuries based on affecting dose andtime (mild moderate and severe) One-third of these victimsare still suffering from SM delayed effects which present anabrupt onset within a period of anywhere from 1 to 40 yearsafter initial exposure [1 2] Medical effects of exposure toSM include ocular skin and respiratory system damage asthe three main areas of concern [1ndash3] furthermore several

studies indicate agitation impression of SM in gastrointesti-nal hematological neuropsychiatric [4] and immune systemeffects [5] and skin cancer increasing [6 7] Patients withdelayed ocularmanifestations of SM suffer from itching pho-tophobia limbal ischemia corneal neovascularization con-junctival thinning and irregularity chronic blepharitis cor-neal degeneration recurring epithelial lesions meibomiangland dysfunction and decreased vision [8ndash10] It is con-siderable that there are significant similarities with knownpathological effects of radiation-induced damages like skinerythema mucositis nausea and diarrhea for acute effectsand fibrosis organ dysfunction necrosis and vascular dam-ages for late effects which are the consequences of animperfect tissue remodeling [11 12]

There are several reports on SM effects and its molec-ular bases in patients The most considered mechanisms

Hindawi Publishing CorporationInternational Journal of ProteomicsVolume 2015 Article ID 659241 10 pageshttpdxdoiorg1011552015659241

2 International Journal of Proteomics

include SM-induced DNA alkylation damage poly(ADP-ribose)polymerase activation calcium signaling and calmod-ulin nitric oxide signaling and oxidative stress [13] inflam-matory reactions matrix-metalloproteinase activity [14]immunologic effects neurotrophic effects and limbal stemcell deficiency [15] Although the phenomena consideredsounds to be similar to the main knownmechanism involvedin radiation-induced late effects (inflammation oxidativedamages fibrosis vascular alterations and cellular depletion)[11 16 17] the underlying pathogenesis and molecular basisof SM acute and delayed effects are still a controversial issue

Ocular surface damages are primarily due to local contactto SM but its systemic toxicity may lead to some serum pro-tein changes that may continually and gradually exacerbatethe ocular surface injuries Here we have analyzed serumproteome of patients against healthy control groups by 2DEfollowed withMALDI TOFTOFmass spectrometry to iden-tify molecular basis of delayed ocular symptoms induced bysevere SM exposure Furthermore we have evaluated several-sample processing approach in order to overcome serumproteomic major challenges such as wide dynamic range ofprotein concentration and presence of high abundance pro-teins like IgG and albumin

2 Materials and Methods

21 Serum Sampling Following institutional ethical approvaland written informed consent blood samples were obtainedfrom ten victims with known pathology of SM severe oculareffects and ten controls by venipuncture into red top tubes(without coagulant activator) All severe victims were manwith average age of 54 plusmn 42 in which nearly 28 plusmn 46 yearshave passed since injury Some of them were treated cornealtransplant surgery and none have any significant pulmonaryand skin complications The blood was centrifuged (for 15minutes at 1200 g in room temperature) immediately to pre-vent secretion interference of blood cells during clottingObtained plasmawas allowed to clot naturally at 4∘C less than60 minutes to prevent protease activities Then the serumwas separated from coagulation factors after centrifugationat 15000 g at 4∘C for 15 minutes Finally the serum samplesthen delipidated by centrifuging at 14000 rpm at room tem-perature for 15min [18] and stored in minus80∘C

22 Albumin and Immunoglobulin Depletion

221 10 Trichloroacetic Acid (TCA)Acetone TCA precipi-tation was carried out as modified method reported by Chenet al [19] 25 120583l of human serum was precipitated by additionof 100 120583l of 10 TCAacetone solution and was mixed bygentle vortexing The mixture was incubated at minus20∘C for90 minutes and then centrifuged at 15000 g and 4∘C for 15minutes Then 1mL of ice-cold acetone (minus20∘C) was addedto wash the precipitate and supernatant was separated Thesample incubated in minus20∘C for 15 minutes and centrifuged asabove The acetone containing supernatant was removed andthe precipitate was dried in air a few minutes

222 ChloroformMethanol This procedure was carried outas reported by Wessel and Flugge [20] One volume serumsample (25 120583l) wasmixedwith four-volumemethanol Simul-taneously by vortex one-volume chloroform and then three-volume double distillate water (DDW) were added and thencentrifuged at 14000 g for 1min and aqueous layer wasremoved Again with vortexing four-volume methanol wasadded and centrifuged at 14000 g for 2min The pellet waswashed by cold acetone (minus20∘C) air-dried for a few minutesthen resolubilized in sample buffer and concentrated

223 Acetone One-volume serum sample was mixed withfour volumes cold acetone (minus20∘C) incubated for 120minin minus20∘C and centrifuged at 15000 g for 15min The pelletwas washed by cold acetone air-dried for a few minutes andconcentrated after resolubilization in sample buffer

224 Ammonium Sulfate One volume of serum was mixedwith two volumes of DDW and 0023 gr ammonium sulfatewas added gradually Incubated about 30min at room tem-perature and then centrifuged at 12000 rpm for 10min Thepellet waswashed by cold acetone and air-dried for a fewmin-utes and concentrated after resolubilization in sample buffer

225 Acetonitrile (ACN) TheACN precipitation was carriedout as reported by Merrell et al [21] with minor modifi-cation One volume of serum was mixed by two volumesof pure ACN They were immediately vortexing for 5 secvigorously incubated for 30min at room temperature andthen centrifuged at 12000 rpm for 10min Upper solutionwas incubated by cold acetone for 60min and centrifugedas above The pellet was resolubilized in sample buffer andconcentrated

226 ProteoMiner Protein Enrichment Kit One volume ofserum sample was performed by ProteoMine Protein Enrich-ment Kit as manual protocol in four steps column prepara-tion binding washing and elution Elution step remainedacidic solution so it was adjusted to pH 74 by PBS bufferthen in order to remove ionic contaminant adjusted to 1mlby cold acetone and incubated for 90min after centrifugationthe pellet was air-dried and resolubilized in sample buffer andconcentrated

23 2DE Precipitated proteins obtained from any of theabove methods were separately dissolved in sample buffer(sodium dodecyl sulfate (SDS) 2wv and dithiothreitol(DTT) 125wv) and their concentration was determinedby UV absorbance at 280 nm (NanoDrop 2000c ThermoScientific NanoDrop Product Wilmington DE USA) Then80 120583g proteins were diluted to 300 120583l by rehydration buffer(urea 7M thiourea 2M CHAPS 4wv ampholyte 3102wv DTT 100mM)The rehydration stage was performedin 17 cm IPG nonlinear pH 3ndash10 strips (Bio-Rad HerculesCA USA) about 16 h actively (constant voltage 50V) Imme-diately isoelectric focusing was performed according to theprogram (i) up to 150V for 90min rapidly (desalting) (ii)focused at 2000V for 180min linearly (iii) up to 10000V

International Journal of Proteomics 3

Table 1 Precipitation methods and ProteoMiner protein enrichment kit efficiency

Precipitation method Protein amount beforeprecipitation (120583g120583L)

Protein amount afterprecipitation (120583g120583L) Percentage of recovery () Number of spots

Acetone 6290 plusmn 367 3696 plusmn 433 5876 plusmn 768 410 plusmn 9ACN 6290 plusmn 367 4402 plusmn 201 6998 plusmn 518 392 plusmn 1110 TCAacetone 6290 plusmn 367 2821 plusmn 321 4485 plusmn 573 438 plusmn 8Ammonium sulphate 6290 plusmn 367 4647 plusmn 406 7388 plusmn 766 375 plusmn 14Chloroformmethanol 6290 plusmn 367 5018 plusmn 267 7978 plusmn 629 357 plusmn 910 TCAacetone 6290 plusmn 367 2821 plusmn 321 4485 plusmn 573 617 plusmn 12lowast

ProteoMiner proteinenrichment kit 6290 plusmn 367 2049 plusmn 316 3258 plusmn 536 576 plusmn 10lowast

Protein concentration was determined by NanoDrop at 280 nm and a number of spots are counted from 7 cm and 17 cm lowastgels by SameSpots Progenesis version33 The results are obtained from six 7 cm and ten 17 cm gels

for 240min linearly (iv) finally maintained at 10000V for atotal of 60000V-h rapidly During isoelectric focusing (IEF)we use wicks before desalting (anode and cathode DDW)and after desalting (anode DDW cathode DTT (20mM))step Then IEF strips were equilibrated for 20 minutes inboth equilibration buffer I (urea 6M Tris-Hcl 15M pH 88SDS 2wv glycerol 20DTT 25wv) and equilibrationbuffer II (urea 6MTris-Hcl 15MpH 88 SDS 2wv glyc-erol 20 iodoacetamide 4wv) respectively The seconddimension was performed on 11 SDS polyacrylamide gels atconstant current ((i) 16mAGel for 30min (ii) 24mAGel)in a Protean II xi 2-D cell (Bio-Rad Hercules CA USA)After protein fixation for overnight withmethanol containingacetic acid and formaldehyde the gel was stained accordingto MS-compatible silver nitrate procedure according to Yanet al [22]

24 Image Processing and Statistical Analysis After staininggels were scanned by GS-800 Calibrated Densitometer (Bio-Rad Hercules CA USA) the scanned gels were analyzedwith SameSpots Progenesis version 33 (and 41) (NonlinearDynamics Durham NC USA) The relative intensities ofspots were used for comparison between chemical warfarevictims and control groups For a comparison of proteinpeak intensity differences between two groups analysis ofvariance (ANOVA) was performed The software providessome powerful multivariate statistics analysis including prin-cipal component analysis (PCA) correlation analysis poweranalysis and 119902-values (false discovery rate adjusted 119875-values)tomake reliable biomarker detection and explore data trendsAlso maximum of coefficients of variance (CV) comparedbetween two groups according to the spots normalizedvolume to explore specific groups of spots that meet chosenstatistical thresholds

25 MALDI TOFTOF and Database Searching The selectedspots were separated fromgelmanually andMS spectrometryanalysis was done at the University of York ldquoSource Bio-Science LifeSciences Englandrdquo Then identification of pro-tein query was performed by using the MASCOT programsearches within ldquoNCBInr 20101130rdquo database according tothe following parameters peptide mass tolerance 100 ppm

MSMS ionmass tolerance 05Da allowing up to onemissedcleavage variable modifications considered were methionineoxidation and cysteine carboxy-amido-methylation

26 Western Blotting Equal amount of depleted serum ofeach groups was pooled and then separated by 11 one-dimensional SDS-PAGE and transferred to nitrocellulosemembranes in a transblot electrophoresis transfer cell (Bio-Rad Hercules CA USA) Western blot analyses were per-formed by using rabbit polyclonal antibodies against Tf(diluted 1 1000 Abcam) and A1AT (diluted 1 500 Sigma-Aldrich) The membranes were blotted with a horserad-ish peroxidase-conjugated goat anti-rabbit IgG secondaryantibody (diluted 1 20000 Sigma-Aldrich) for 1 h and thendetected with enhanced chemiluminescence reagents for1min Band intensities were quantified using ldquoGelQuantNETsoftware provided by httpwwwbiochemlabsolutionscomrdquo

3 Results

31 Sample Preparation Among precipitation methods 10TCAacetone indicates better 2D pattern according to theremoval of albumin and IgG (Table 1) Also this precipitationmethod in comparison with ProteoMiner Kit shows morespot numbers while ProteoMiner Kit removes albumin andIgG almost completely leading to nonspecific removal ofsome proteins and minor spot number (Figure 1)

32 Image Processing and Protein Spots Detection Thequalityof grouping was evaluated by PCA plot for spots with ldquoanova119875 value lt 005rdquo (Figure 2(a)) as recommended Comparisonof principal components 1 and 2 when all spots were selected(2686 and 1366 resp) against when only significantspots with 119875 value lt 05 were selected (5674 and 969resp) clearly indicates that the groups are more stronglyseparated in PCA plot Analysis of power was performedaccording to recommended threshold for that adjusted atpower gt 008 among selected spots with ANOVA 119875 valuelt 005 (Figure 2(b)) This diagram indicates at least ninereplicates needed to achieving 80 confidence in the exper-imental data to find the differences that do actually existSimilarity in expression profile of selected spotswas evaluated


+ minus

(a)

+ minus

80768kD

54953kD

13800 kD

(b)

Figure 1 2DE gel comparison 10TCAacetone (b) and ProteoMine large-Capacity Kit (a)Molecular weight assigned by software accordingto intrinsic standards and verified according to identical proteins in SWISS-2DPAGE database

03

02

01

00

minus01

minus02minus010 minus005 000 005 010 015

Principal components 1

Principal components analysis

Prin

cipa

l com

pone

nts2

(a)

10090807060504030201001 2 3 4 5 6 7 8 9 10 11

Number of replicates

Power analysis

Spot

s with

pow

ergt08

()

(b)

Figure 2 Multivariate statistics (a) Principal component analysis (PCA) plots for spots with ANOVA 119875 value lt 005 Principal components1 and 2 are 5674 and 969 respectively Control (circle) patient (triangle) (b) Power analysis diagram according to those spots withANOVA 119875 value lt 005

using correlation analysis (Figure 3) The obtained dendro-gram reveals that except six spots with downregulation char-acteristic in victims rest of them represent upregulationvariation in expression profile

Overall with software analysis 617 spots were detectedon silver stained pattern in this study Among them 102spots were tagged with ANOVA 119875 value lt 005 67 spotswere tagged with fold ge2 and 42 spots were tagged withpower gt08 Eventually result of multivariate statistics leadsto selection of twenty spots with both ANOVA 119875 value lt005 and power ge 08 In final stage thirteen spots (exceptkeratin 1) were identified by using MALDI TOFTOF massspectrometry and search within NCBI database with MAS-COT (Table 2)

33 Validation by Western Blotting To confirm the massspectrometry obtained data we performed western blotanalyses of two proteins Tf andA1AT as shown in Figure 4(c)The immunoblot analysis also showed decreased A1AT andTf isoforms in the pooled serum of severe patient comparedwith healthy control group

4 Discussion

According to mentioned approach we identified thirteenproteins including albumin keratin (except keratin 1) andHp isoforms as well as immunoglobulin kappa chain thatwere upregulated in patients plus two downregulated proteinsincluding Tf and A1AT

41 Investigation of 2DE Method Our finding showed thatChen et alrsquos [19] procedure eventuates the best quality of two-dimensional gel profile in comparison with relevant availableprecipitation protocols In addition further modificationof chenrsquos method indicated more protein spots as well asless streaking in 2DE profile than ProteoMiner proteinenrichment Kit (Figure 1) TCAacetone precipitation hasshown good potential for minimizing protein degradation byproteases and removal of contaminant such as salts [23]

42 Hp All identified Hp isoforms showed upregulation inour study (Figure 4(a)) Hp is a plasma 120572

2-glyco-protein that

belongs to the family of acute phase proteins [24] The Hp-1


Dendrogram

Dist

ance

20

15

10

05

00

1063

1313

1330

1331

1344

1377

1458

1466

1469

1481

1482

1490

1487

1524

1495

1558

1625

1662

2168

2487

2563

2570

2758

2796

2828

2837

2847

2888

2914

2921

3026

3107

3120

3139

3249

ID of selected spots

4

3

2

1

0

minus1

minus2

minus3

4

3

2

1

0

minus1

minus2

minus3

Stan

dard

ised

norm

alise

d vo

lum

Stan

dard

ised

norm

alise

d vo

lum

Figure 3 Correlation analysis dendrogram for final selected spots indicates similarity in their expression profiles Six spots indicated in leftof dendrogram showing downregulation in patient while others are upregulated

and Hp-2 have the same 120573 chain but they differ in their 120572chain isoforms [25]

There are several well-known roles for Hp but the moresignificant of them is as hemoglobin (Hb) binding proteinTheir complex serves to prevent the oxidation damages [2627] Iron derived from Hb can catalyze a series of oxidativereaction that is able to participate in organic and inorganicoxygen radical reactions and promoting the accumulationof hydroxyl radicals according to Fenton and Heber-Weissreactions [28 29] Also reactive oxygen and nitrogen spacesseem to contribute as key elements in the radiation-inducedlate effects which could promote the fibrogenesis throughcontribution in the transforming growth factor-120573 (TGF120573)signaling pathway activation [11 17] It is notable that thecomplexes of Hp-2Hb have a higher half-life in the circu-lation than Hp-1Hb complexes causing increased accumu-lation of Hb in the plasma and the tissues particularly in theproximal tubule of the kidney resulting in vascular damages[30] Hp plays a double-edge role While its expression is

elevated to prevent oxidative stress of Hb derived iron viastabilizing heme in the heme pocket of Hb increasing of Hp-2 genotype due to longer half-life of its Hp-Hb complexesin circulation leads to increased accumulation of Hb and itsderived iron in the plasma and the tissues Many authorsreviewed free iron oxidative effects in human health [31ndash34]Also the allele frequencies of Hp and their association indiseases are reviewed in detail by Carter and Worwood [35]

AlreadyMehrani et al foundHp isoforms overexpressionin plasma of lung disease patient whom exposed by SM [36]They concluded ongoing tissue remodeling is involved inSM exposed lung damage patients In addition they foundHp isoforms in bronchoalveolar lavage fluid of SM exposedpatients were significantly elevated in moderate and severelung disease patients compared to mild and healthy controls[37] de Kleijn et al have shown that arterial expressed Hpstimulates angiogenesis tissue regeneration and cell migra-tion through inducing accumulation of gelatin by inhibitiongelatinases such as MMP-2 and MMP-9 [38] These collagen


(A)

(A998400)

(a)

(A)

(A998400)

Tf A1AT

(b)

b1 b2

b3 b4

Patient Control

Tf

A1AT

(A) (B)

(c)

Figure 4 Protein expression profile (a) Different spots of serum haptoglobin (Hp) isoforms that are identified by MALDI TOFTOF Upper(A) and lower (A1015840) rows refer to patients and controls respectively (b) Transferrin (Tf) and alpha 1 antitrypsin (A1AT) Upper (A) and lower(A1015840) rows refer to patient and control respectively (c) (A) Western blot validation of transferrin (Tf) and alpha 1 antitrypsin (A1AT) (B)Intensity profile obtained with GelQuantNET software provided by httpwwwbiochemlabsolutionscom Background signal is 782907395and band signals are 14578867 in b1 20306096 in b2 1114966 in b3 and 15347117 in b4 respectively


Table2MALD

ITOFTO

FMSspectro

metry

proteinidentifi

catio

nin

2DEmap

ofserum

Proteinname

Accessionnu

mber

Expressio

nlevellowast

Biologicalfunctio

n119875value119902value

Power

Fold

Max

CVdagger

MASC

OT

proteinscore

Sequ

ence

coverage

()

PIDagger

Mw(D

a)para

Keratin

1gi|7331218

uarrStructuralproteins

000

079

004

099

2298

121

4816

66149

Keratin

10gi|18

6629

uarr000

079

004

099

2298

1145

472

39832

Cytokeratin

9gi|435476

uarr000

444

009

091

152634

174

5519

62320

Keratin

1gi|7331218

uarr000

444

009

091

152634

785

16816

66149

Serum

albu

min

gi|28592

uarr

Molecular

carriera

ndregu

latoro

fosm

otic

pressure

inbloo

d000

678

011

086

193642

264

6605

71316

Serum

albu

min

gi|28592

uarr000519

012

089

21

3832

149

4605

71316

Serum

albu

min

gi|28592

uarr000284

007

094

182876

308

9605

71316

Serum

albu

min

gi|28592

uarr000740

011

085

183143

632

605

71316

Haptoglob

inhp

2gi|223976

uarr

Free

hemoglobinbind

ing

proteinin

plasma(

iron

oxidativea

ctivity

supp

ressor)

000105

004

098

28

4480

115

6623

42344

Haptoglob

inhp

2gi|223976

uarr000372

008

092

23

4011

703

623

42344

Haptoglob

inhp

2gi|223976

uarr000705

011

086

32

4662

139

6623

42344

Haptoglob

inhp

2gi|223976

uarr000231

006

095

35

7920

279

11623

42344

Igkapp

agi|10

6529

uarrIm

mun

oglobu

lin000511

010

089

183777

5416

561

11161

Transfe

rrin

gi|339469

darrIron

transport

000

979

013

082

152225

503

840

80768

alph

a1antitrypsin

gi|17

7831

darrprotease

inhibitor

000159

005

097

192626

584

491

57706

Max

CVischosen

bycomparis

onso

fCVam

ongcontroland

patie

ntgrou

pslowastexpressio

nlevelsho

wsp

rotein

changesinthep

atient

incomparis

onwith

controlsdaggerMaxim

umof

coeffi

ciento

fvarianceo

btained

incomparis

onbetweencontroland

patie

ntgrou

pDaggerIsoelectric

point

para Molecular

weightinDalton


deposition and extracellular matrix accumulation have beenalso due to activation of TGF120573 signaling pathway after radi-ation exposure [11] From this point of view our findingcould elevate the HP and MMP roles in delayed SM ocularpathology which are also previously discussed by Sardasht-Iran cohort studies in long-term pulmonary complicationinduced by SM [39 40]

43 Tf In our study Tf has been downregulated(Figure 4(b)) Tf is a plasma glycoprotein which has thecapacity to bind to two free atoms of ferric iron (Fe3+) inbiological fluid [41]

Tf represents a protectivemechanismagainst the presenceof free iron in the plasma [42] which could be extremelytoxicThe increasing level of serumTf leads to iron deficiencyanemia while decreasing level of that leads to iron overloaddisorders [31 43 44] The level of Tf also decreases duringinflammation infection and malignancy conditions [45]Therefore reduction of Tf level as we found could leadto imbalances in free iron homeostasis effects includingfree radical production mutation and also cell damagesFurthermore Tf deficiency anemia could induce expressionof transcription factors such as hypoxia induced factors dueto lack of oxygen Hypoxia promotes the formation of bloodvessels and induces angiogenesis process [46] This tissuehypoxia could seemingly be one of the mechanisms perpetu-ating the fibrogenic response in radiation-induced late effectsdue to vascular damage and tissue remodeling [11 17]

44 A1AT This protein also revealed downregulation in ourstudy (Figure 4(b))whileYarmohammadi et al have reportedsignificantly higher amount of salivary A1AT in salivary ofsulfur mustard exposed patients 20 years after the exposure[47]

A1AT like Hp is an acute phase glycoprotein and pro-totypic member of the Serpin family [48 49] It has awide spectrum of antiprotease activity and inhibits severalserine proteases such as neutrophil elastase cathepsin-Gand proteinase-3 [50] It thought to play an importantrole in limiting host tissue injury by proteases at sites ofinflammation [49] Several studies have shown that A1ATspecifically inhibits the activity of caspase-3 [51ndash53] A1ATdeficiency is the most widely recognized abnormality of aproteinase inhibitor The inherited A1AT deficiency exhibitsan increased susceptibility to chronic inflammatory condi-tions including chronic obstructive lung disease liver dis-eases and occasionally systemic vasculitis and necrotizingpanniculitis [54] Here it appears that lack of A1AT leads toprotease induced tissue damages which cooperate withMMPactivity eventuating cell apoptosis which could promote inassociation with free iron toxicity [52 53 55]

Findings that A1AT enhances the synthesis of bothtransferrin receptor and ferritin revealed a role of A1AT iniron metabolism [48] Here it would be considered maybethat A1AT reduction affects incompetency of Tf receptor andferritin syntheses and lead to Hp over expression due tocytokines (TNF-120572) stimulation effects [48 56 57] Also thesecytokines showed a significant increase in radiation-inducedchronic inflammation [12]

5 Conclusions

Our goal was to find a molecular basis for sulfur mus-tard delayed ocular symptoms and eventually treatment ofpatients Finally our finding elevated important effects of freeiron induced oxidative stresses which are supplemented withprotease activity (especiallyMMP) in development of delayedeye symptoms which both indirectly could uphold celldepletion supposition as bases for delayed ocular symptomsAlthough the rules of all mentioned proteins are not specifiedhere completely delayed ocular injuries may be partiallyexplained by these protein changes Also it can be inferredthat changes in serum constituents are due to systemic sideeffects of SM rather than its local ocular side effects We hopethat our results have revealed some furtive aspects of SMexposed delayed ocular symptoms that will be promoted byfuture studies

Abbreviations

ACN AcetonitrileA1AT Alpha 1 antitrypsinANOVA Analysis of varianceDTT DithiothreitolDDW Double distillate waterHp HaptoglobinHb HemoglobinIEF Isoelectric focusingMALDI-TOFTOF Matrix-assisted laser

desorptionionizationtime-of-flighttime-of-flight

MMP Matrix-metalloproteinasePCA Principal component analysisSDS Sodium dodecyl sulfateSM Sulfur mustardTf TransferrinTGF120573 Transforming growth factor-120573TCA Trichloroacetic acidTNF-120572 Tumor necrosis factor alpha2DE Two-dimensional electrophoresis

Ethical Approval

The study was performed in accordance with the Declarationof Helsinki and was approved by the Institutional ReviewBoard of Tarbiat Modares University Tehran Iran

Conflict of Interests

The authors have declared no conflict of interests

Authorsrsquo Contribution

Zaiddodine Pashandi participated in the design of the studycarried out all experiments and prepared paper draft NedaSaraygord-Afshari participated in the design of the studyand software analysis and helped to draft the paper HosseinNaderi-Manesh participated in the design of the study andreviewed and prepared the final paper Mostafa Naderi


participated in patient selection and sampling All authorsread and approved the final paper

Acknowledgment

This work was supported by the Tarbiat Modares University

References

[1] K Kehe and L Szinicz ldquoMedical aspects of sulphur mustardpoisoningrdquo Toxicology vol 214 no 3 pp 198ndash209 2005

[2] M Rowell K Kehe F Balszuweit and H Thiermann ldquoThechronic effects of sulfurmustard exposurerdquo Toxicology vol 263no 1 pp 9ndash11 2009

[3] M Wattana and T Bey ldquoMustard gas or sulfur mustard anold chemical agent as a new terrorist threatrdquo Prehospital andDisaster Medicine vol 24 no 1 pp 19ndash31 2009

[4] E Darchini-Maragheh H Nemati-Karimooy H Hasanabadiand M Balali-Mood ldquoDelayed Neurological Complications ofSulphur Mustard and Tabun Poisoning in 43 Iranian VeteransrdquoBasic and Clinical Pharmacology and Toxicology vol 111 no 6pp 426ndash432 2012

[5] M Balali-Mood and M Hefazi ldquoComparison of early and latetoxic effects of sulfur mustard in Iranian veteransrdquo Basic ampClinical Pharmacology amp Toxicology vol 99 no 4 pp 273ndash2822006

[6] M Balali-Mood M Hefazi M Mahmoudi et al ldquoLong-term complications of sulphur mustard poisoning in severelyintoxicated Iranian veteransrdquo Fundamental and Clinical Phar-macology vol 19 no 6 pp 713ndash721 2005

[7] M Shohrati M Davoudi M Ghanei M Peyman and APeyman ldquoCutaneous and ocular late complications of sulfurmustard in Iranian veteransrdquo Cutaneous and Ocular Toxicologyvol 26 no 2 pp 73ndash81 2007

[8] S Khateri M Ghanei S KeshavarzM Soroush andD HainesldquoIncidence of lung eye and skin lesions as late complicationsin 34000 Iranians with wartime exposure to mustard agentrdquoJournal of Occupational and Environmental Medicine vol 45no 11 pp 1136ndash1143 2003

[9] H Ghasemi T Ghazanfari M Babaei et al ldquoLong-term ocularcomplications of sulfur mustard in the civilian victims ofSardasht Iranrdquo Cutaneous and Ocular Toxicology vol 27 no 4pp 317ndash326 2008

[10] H Ghasemi T Ghazanfari M Ghassemi-Broumand et alldquoLong-term ocular consequences of sulfur mustard in seriouslyeye-injured war veteransrdquo Cutaneous and Ocular Toxicologyvol 28 no 2 pp 71ndash77 2009

[11] S M Bentzen ldquoPreventing or reducing late side effects ofradiation therapy radiobiology meets molecular pathologyrdquoNature Reviews Cancer vol 6 no 9 pp 702ndash713 2006

[12] P Gallet B Phulpin J-L Merlin et al ldquoLong-term alterationsof cytokines and growth factors expression in irradiated tissuesand relation with histological severity scoringrdquo PLoS ONE vol6 no 12 Article ID e29399 2011

[13] K Kehe F Balszuweit D Steinritz and HThiermann ldquoMolec-ular toxicology of sulfur mustard-induced cutaneous inflam-mation and blisteringrdquo Toxicology vol 263 no 1 pp 12ndash192009

[14] K Kehe F Balszuweit J Emmler H Kreppel M Jochumand H Thiermann ldquoSulfur mustard researchmdashstrategies for

the development of improved medical therapyrdquo Eplasty vol 8article e32 2008

[15] Y Panahi M Naderi M A Zare and Z Poursaleh ldquoOculareffects of sulfurmustardrdquo Iranian Journal of Ophthalmology vol25 no 2 pp 90ndash106 2013

[16] H B Stone C N Coleman M S Anscher and W HMcBride ldquoEffects of radiation on normal tissue consequencesand mechanismsrdquo Lancet Oncology vol 4 no 9 pp 529ndash5362003

[17] H P Rodemann and M A Blaese ldquoResponses of normal cellsto ionizing radiationrdquo Seminars in Radiation Oncology vol 17no 2 pp 81ndash88 2007

[18] R Meng M Gormley V B Bhat A Rosenberg and A AQuong ldquoLow abundance protein enrichment for discovery ofcandidate plasma protein biomarkers for early detection ofbreast cancerrdquo Journal of Proteomics vol 75 no 2 pp 366ndash3742011

[19] Y-Y Chen S-Y Lin Y-Y Yeh et al ldquoA modified proteinprecipitation procedure for efficient removal of albumin fromserumrdquo Electrophoresis vol 26 no 11 pp 2117ndash2127 2005

[20] D Wessel and U I Flugge ldquoA method for the quantitativerecovery of protein in dilute solution in the presence of deter-gents and lipidsrdquoAnalytical Biochemistry vol 138 no 1 pp 141ndash143 1984

[21] K Merrell K Southwick S W Graves M Sean Esplin NE Lewis and C D Thulin ldquoAnalysis of low-abundance low-molecular-weight serum proteins using mass spectrometryrdquoJournal of Biomolecular Techniques vol 15 no 4 pp 238ndash2482004

[22] J X Yan R Wait T Berkelman et al ldquoA modified silverstaining protocol for visualization of proteins compatible withmatrix-assisted laser desorptionionization and electrosprayionization-mass spectrometryrdquo Electrophoresis vol 21 no 17pp 3666ndash3672 2000

[23] A Gorg W Weiss and M J Dunn ldquoCurrent two-dimensionalelectrophoresis technology for proteomicsrdquo Proteomics vol 4no 12 pp 3665ndash3685 2004

[24] I K Quaye ldquoHaptoglobin inflammation and diseaserdquo Transac-tions of the Royal Society of Tropical Medicine and Hygiene vol102 no 8 pp 735ndash742 2008

[25] F Polticelli A Bocedi G Minervini and P Ascenzi ldquoHumanhaptoglobin structure and functionmdasha molecular modellingstudyrdquo FEBS Journal vol 275 no 22 pp 5648ndash5656 2008

[26] M Haase R Bellomo and A Haase-Fielitz ldquoNovel biomarkersoxidative stress and the role of labile iron toxicity in cardiopul-monary bypass-associated acute kidney injuryrdquo Journal of theAmerican College of Cardiology vol 55 no 19 pp 2024ndash20332010

[27] H Goldenstein N S Levy and A P Levy ldquoHaptoglobingenotype and its role in determining heme-iron mediatedvascular diseaserdquo Pharmacological Research vol 66 no 1 pp1ndash6 2012

[28] SMH Sadrzadeh E Graf S S Panter P E Hallaway and JWEaton ldquoHemoglobin A biologic Fenton reagentrdquoThe Journal ofBiological Chemistry vol 259 no 23 pp 14354ndash14356 1984

[29] J M Rifkind S Ramasamy P T Manoharan E Nagababu andJ G Mohanty ldquoRedox reactions of hemoglobinrdquo Antioxidantsand Redox Signaling vol 6 no 3 pp 657ndash666 2004

[30] L Marquez C Shen I Cleynen et al ldquoEffects of haptoglobinpolymorphisms and deficiency on susceptibility to inflamma-tory bowel disease and on severity of murine colitisrdquo Gut vol61 no 4 pp 528ndash534 2012


[31] R E Fleming and P Ponka ldquoIron overload in human diseaserdquoThe New England Journal of Medicine vol 366 no 4 pp 348ndash359 2012

[32] M W Hentze M U Muckenthaler and N C Andrews ldquoBal-ancing acts molecular control ofmammalian ironmetabolismrdquoCell vol 117 no 3 pp 285ndash297 2004

[33] G Papanikolaou and K Pantopoulos ldquoIron metabolism andtoxicityrdquo Toxicology and Applied Pharmacology vol 202 no 2pp 199ndash211 2005

[34] S Johnson ldquoIron catalyzed oxidative damage in spite ofnormal ferritin and transferrin saturation levels and its possiblerole in Wernerrsquos syndrome Parkinsonrsquos disease cancer goutrheumatoid arthritis etcrdquoMedical Hypotheses vol 55 no 3 pp242ndash244 2000

[35] K Carter and M Worwood ldquoHaptoglobin a review of themajor allele frequencies worldwide and their association withdiseasesrdquo International Journal of Laboratory Hematology vol29 no 2 pp 92ndash110 2007

[36] H Mehrani M Ghanei J Aslani and Z Tabatabaei ldquoPlasmaproteomic profile of sulfur mustard exposed lung diseasespatients using 2-dimensional gel electrophoresisrdquo Clinical Pro-teomics vol 8 no 1 article 2 2011

[37] H Mehrani M Ghanei J Aslani and L Golmanesh ldquoBron-choalveolar lavage fluid proteomic patterns of sulfur mustard-exposed patientsrdquo Proteomics Clinical Applications vol 3 no10 pp 1191ndash1200 2009

[38] D P V de Kleijn M B Smeets P P C W Kemmerenet al ldquoAcute-phase protein haptoglobin is a cellmigration factorinvolved in arterial restructuringrdquo The FASEB Journal vol 16no 9 pp 1123ndash1125 2002

[39] S Pourfarzam R Yaraee Z M Hassan et al ldquoChemokinesMMP-9 and PMN elastase in spontaneous sputum of sulfurmustard exposed civilians sardasht-Iran Cohort Studyrdquo Inter-national Immunopharmacology vol 17 no 3 pp 958ndash963 2013

[40] A Kiani A Mostafaie F H Shirazi and T GhazanfarildquoSerum profiles of matrix metalloproteinases and their tissueinhibitors in long-term pulmonary complication induced bysulfur mustard Sardasht-Iran Cohort Study (SICS)rdquo Interna-tional Immunopharmacology vol 17 no 3 pp 964ndash967 2013

[41] R R Crichton and M Charloteaux-Wauters ldquoIron transportand storagerdquo European Journal of Biochemistry vol 164 no 3pp 485ndash506 1987

[42] D Szoke and M Panteghini ldquoDiagnostic value of transferrinrdquoClinica Chimica Acta vol 413 no 15-16 pp 1184ndash1189 2012

[43] P BrissotM Ropert C Le Lan andO Loreal ldquoNon-transferrinbound iron a key role in iron overload and iron toxicityrdquoBiochimica et Biophysica Acta General Subjects vol 1820 no3 pp 403ndash410 2012

[44] A Taher K M Musallam F El Rassi et al ldquoLevels of non-transferrin-bound iron as an index of iron overload in patientswith thalassaemia intermediardquo British Journal of Haematologyvol 146 no 5 pp 569ndash572 2009

[45] R F Ritchie G E Palomaki L M Neveux O NavolotskaiaT B Ledue and W Y Craig ldquoReference distributions forthe negative acute-phase serum proteins albumin transferrinand transthyretin a practical simple and clinically relevantapproach in a large cohortrdquo Journal of Clinical LaboratoryAnalysis vol 13 no 6 pp 273ndash279 1999

[46] J Harned J Ferrell S NagarMGoralska L N Fleisher andMC McGahan ldquoCeruloplasmin alters intracellular iron regulatedproteins and pathways ferritin transferrin receptor glutamate

and hypoxia-inducible factor-1120572rdquo Experimental Eye Researchvol 97 no 1 pp 90ndash97 2012

[47] M E Yarmohammadi Z M Hassan A Mostafaie et al ldquoSali-vary levels of secretary IgAC5a and alpha 1-antitrypsin in sulfurmustard exposed patients 20 years after the exposure Sardasht-IranCohort Study (SICS)rdquo International Immunopharmacologyvol 17 no 3 pp 952ndash957 2013

[48] S M Janciauskiene R Bals R Koczulla C Vogelmeier TKohnlein and T Welte ldquoThe discovery of 1205721-antitrypsin andits role in health and diseaserdquo Respiratory Medicine vol 105 no8 pp 1129ndash1139 2011

[49] D A Bergin K Hurley N G McElvaney and E P ReevesldquoAlpha-1 antitrypsin a potent anti-inflammatory and potentialnovel therapeutic agentrdquo Archivum Immunologiae et TherapiaeExperimentalis vol 60 no 2 pp 81ndash97 2012

[50] J Duranton and J G Bieth ldquoInhibition of proteinase 3 by[alpha]1-antitrypsin in vitro predicts very fast inhibition invivordquo The American Journal of Respiratory Cell and MolecularBiology vol 29 no 1 pp 57ndash61 2003

[51] L Shapiro G B Pott and A H Ralston ldquoAlpha-1-antitrypsininhibits human immunodeficiency virus type 1rdquo The FASEBJournal vol 15 no 1 pp 115ndash122 2001

[52] I Petrache I Fijalkowska T R Medler et al ldquo120572-1 antitrypsininhibits caspase-3 activity preventing lung endothelial cellapoptosisrdquo The American Journal of Pathology vol 169 no 4pp 1155ndash1166 2006

[53] B Zhang Y Lu M Campbell-Thompson et al ldquo1205721-antitrypsinprotects 120573-cells from apoptosisrdquo Diabetes vol 56 no 5 pp1316ndash1323 2007

[54] J K Stoller and L S Aboussouan ldquoAlpha1-antitrypsin defi-ciencyrdquoThe Lancet vol 365 no 9478 pp 2225ndash2236 2005

[55] R Ray C M Simbulan-Rosenthal B M Keyser et al ldquoSulfurmustard induces apoptosis in lung epithelial cells via a caspaseamplification looprdquo Toxicology vol 271 no 3 pp 94ndash99 2010

[56] A P Levy R Asleh S Blum et al ldquoHaptoglobin basic andclinical aspectsrdquo Antioxidants and Redox Signaling vol 12 no2 pp 293ndash304 2010

[57] S M Janciauskiene I M Nita and T Stevens ldquoAlpha1-antitrypsin old dog new tricks 1205721- antitrypsin exerts in vitroanti-inflammatory activity in human monocytes by elevatingcAMPrdquoThe Journal of Biological Chemistry vol 282 no 12 pp8573ndash8582 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


include SM-induced DNA alkylation damage poly(ADP-ribose)polymerase activation calcium signaling and calmod-ulin nitric oxide signaling and oxidative stress [13] inflam-matory reactions matrix-metalloproteinase activity [14]immunologic effects neurotrophic effects and limbal stemcell deficiency [15] Although the phenomena consideredsounds to be similar to the main knownmechanism involvedin radiation-induced late effects (inflammation oxidativedamages fibrosis vascular alterations and cellular depletion)[11 16 17] the underlying pathogenesis and molecular basisof SM acute and delayed effects are still a controversial issue

Ocular surface damages are primarily due to local contactto SM but its systemic toxicity may lead to some serum pro-tein changes that may continually and gradually exacerbatethe ocular surface injuries Here we have analyzed serumproteome of patients against healthy control groups by 2DEfollowed withMALDI TOFTOFmass spectrometry to iden-tify molecular basis of delayed ocular symptoms induced bysevere SM exposure Furthermore we have evaluated several-sample processing approach in order to overcome serumproteomic major challenges such as wide dynamic range ofprotein concentration and presence of high abundance pro-teins like IgG and albumin

2 Materials and Methods

21 Serum Sampling Following institutional ethical approvaland written informed consent blood samples were obtainedfrom ten victims with known pathology of SM severe oculareffects and ten controls by venipuncture into red top tubes(without coagulant activator) All severe victims were manwith average age of 54 plusmn 42 in which nearly 28 plusmn 46 yearshave passed since injury Some of them were treated cornealtransplant surgery and none have any significant pulmonaryand skin complications The blood was centrifuged (for 15minutes at 1200 g in room temperature) immediately to pre-vent secretion interference of blood cells during clottingObtained plasmawas allowed to clot naturally at 4∘C less than60 minutes to prevent protease activities Then the serumwas separated from coagulation factors after centrifugationat 15000 g at 4∘C for 15 minutes Finally the serum samplesthen delipidated by centrifuging at 14000 rpm at room tem-perature for 15min [18] and stored in minus80∘C

22 Albumin and Immunoglobulin Depletion

221 10 Trichloroacetic Acid (TCA)Acetone TCA precipi-tation was carried out as modified method reported by Chenet al [19] 25 120583l of human serum was precipitated by additionof 100 120583l of 10 TCAacetone solution and was mixed bygentle vortexing The mixture was incubated at minus20∘C for90 minutes and then centrifuged at 15000 g and 4∘C for 15minutes Then 1mL of ice-cold acetone (minus20∘C) was addedto wash the precipitate and supernatant was separated Thesample incubated in minus20∘C for 15 minutes and centrifuged asabove The acetone containing supernatant was removed andthe precipitate was dried in air a few minutes

222 ChloroformMethanol This procedure was carried outas reported by Wessel and Flugge [20] One volume serumsample (25 120583l) wasmixedwith four-volumemethanol Simul-taneously by vortex one-volume chloroform and then three-volume double distillate water (DDW) were added and thencentrifuged at 14000 g for 1min and aqueous layer wasremoved Again with vortexing four-volume methanol wasadded and centrifuged at 14000 g for 2min The pellet waswashed by cold acetone (minus20∘C) air-dried for a few minutesthen resolubilized in sample buffer and concentrated

223 Acetone One-volume serum sample was mixed withfour volumes cold acetone (minus20∘C) incubated for 120minin minus20∘C and centrifuged at 15000 g for 15min The pelletwas washed by cold acetone air-dried for a few minutes andconcentrated after resolubilization in sample buffer

224 Ammonium Sulfate One volume of serum was mixedwith two volumes of DDW and 0023 gr ammonium sulfatewas added gradually Incubated about 30min at room tem-perature and then centrifuged at 12000 rpm for 10min Thepellet waswashed by cold acetone and air-dried for a fewmin-utes and concentrated after resolubilization in sample buffer

225 Acetonitrile (ACN) TheACN precipitation was carriedout as reported by Merrell et al [21] with minor modifi-cation One volume of serum was mixed by two volumesof pure ACN They were immediately vortexing for 5 secvigorously incubated for 30min at room temperature andthen centrifuged at 12000 rpm for 10min Upper solutionwas incubated by cold acetone for 60min and centrifugedas above The pellet was resolubilized in sample buffer andconcentrated

226 ProteoMiner Protein Enrichment Kit One volume ofserum sample was performed by ProteoMine Protein Enrich-ment Kit as manual protocol in four steps column prepara-tion binding washing and elution Elution step remainedacidic solution so it was adjusted to pH 74 by PBS bufferthen in order to remove ionic contaminant adjusted to 1mlby cold acetone and incubated for 90min after centrifugationthe pellet was air-dried and resolubilized in sample buffer andconcentrated

23 2DE Precipitated proteins obtained from any of theabove methods were separately dissolved in sample buffer(sodium dodecyl sulfate (SDS) 2wv and dithiothreitol(DTT) 125wv) and their concentration was determinedby UV absorbance at 280 nm (NanoDrop 2000c ThermoScientific NanoDrop Product Wilmington DE USA) Then80 120583g proteins were diluted to 300 120583l by rehydration buffer(urea 7M thiourea 2M CHAPS 4wv ampholyte 3102wv DTT 100mM)The rehydration stage was performedin 17 cm IPG nonlinear pH 3ndash10 strips (Bio-Rad HerculesCA USA) about 16 h actively (constant voltage 50V) Imme-diately isoelectric focusing was performed according to theprogram (i) up to 150V for 90min rapidly (desalting) (ii)focused at 2000V for 180min linearly (iii) up to 10000V













3 Results




+ minus

(a)

+ minus

80768kD

54953kD

13800 kD

(b)


03

02

01

00

minus01




Prin

cipa

l com

pone

nts2

(a)

10090807060504030201001 2 3 4 5 6 7 8 9 10 11


Power analysis

Spot

s with

pow

ergt08

()

(b)





4 Discussion







Dendrogram

Dist

ance

20

15

10

05

00

1063

1313

1330

1331

1344

1377

1458

1466

1469

1481

1482

1490

1487

1524

1495

1558

1625

1662

2168

2487

2563

2570

2758

2796

2828

2837

2847

2888

2914

2921

3026

3107

3120

3139

3249


4

3

2

1

0

minus1

minus2

minus3

4

3

2

1

0

minus1

minus2

minus3

Stan

dard

ised

norm

alise

d vo

lum

Stan

dard

ised

norm

alise

d vo

lum







(A)

(A998400)

(a)

(A)

(A998400)

Tf A1AT

(b)

b1 b2

b3 b4

Patient Control

Tf

A1AT

(A) (B)

(c)



Table2MALD

ITOFTO

FMSspectro

metry

proteinidentifi

catio

nin

2DEmap

ofserum

Proteinname

Accessionnu

mber

Expressio

nlevellowast

Biologicalfunctio


Power

Fold

Max

CVdagger

MASC

OT

proteinscore

Sequ

ence

coverage

()

PIDagger

Mw(D

a)para

Keratin

1gi|7331218


000

079

004

099

2298

121

4816

66149

Keratin

10gi|18

6629

uarr000

079

004

099

2298

1145

472

39832

Cytokeratin

9gi|435476

uarr000

444

009

091

152634

174

5519

62320

Keratin

1gi|7331218

uarr000

444

009

091

152634

785

16816

66149

Serum

albu

min

gi|28592

uarr

Molecular

carriera

ndregu

latoro

fosm

otic

pressure

inbloo

d000

678

011

086

193642

264

6605

71316

Serum

albu

min

gi|28592

uarr000519

012

089

21

3832

149

4605

71316

Serum

albu

min

gi|28592

uarr000284

007

094

182876

308

9605

71316

Serum

albu

min

gi|28592

uarr000740

011

085

183143

632

605

71316

Haptoglob

inhp

2gi|223976

uarr

Free

hemoglobinbind

ing

proteinin

plasma(

iron

oxidativea

ctivity

supp

ressor)

000105

004

098

28

4480

115

6623

42344

Haptoglob

inhp

2gi|223976

uarr000372

008

092

23

4011

703

623

42344

Haptoglob

inhp

2gi|223976

uarr000705

011

086

32

4662

139

6623

42344

Haptoglob

inhp

2gi|223976

uarr000231

006

095

35

7920

279

11623

42344

Igkapp

agi|10

6529

uarrIm

mun

oglobu

lin000511

010

089

183777

5416

561

11161

Transfe

rrin

gi|339469

darrIron

transport

000

979

013

082

152225

503

840

80768

alph

a1antitrypsin

gi|17

7831

darrprotease

inhibitor

000159

005

097

192626

584

491

57706

Max

CVischosen

bycomparis

onso

fCVam

ongcontroland

patie

ntgrou

pslowastexpressio

nlevelsho

wsp

rotein

changesinthep

atient

incomparis

onwith

controlsdaggerMaxim

umof

coeffi

ciento

fvarianceo

btained

incomparis

onbetweencontroland

patie

ntgrou

pDaggerIsoelectric

point

para Molecular

weightinDalton








5 Conclusions


Abbreviations




Ethical Approval








Acknowledgment


References




































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













3 Results




+ minus

(a)

+ minus

80768kD

54953kD

13800 kD

(b)


03

02

01

00

minus01




Prin

cipa

l com

pone

nts2

(a)

10090807060504030201001 2 3 4 5 6 7 8 9 10 11


Power analysis

Spot

s with

pow

ergt08

()

(b)





4 Discussion







Dendrogram

Dist

ance

20

15

10

05

00

1063

1313

1330

1331

1344

1377

1458

1466

1469

1481

1482

1490

1487

1524

1495

1558

1625

1662

2168

2487

2563

2570

2758

2796

2828

2837

2847

2888

2914

2921

3026

3107

3120

3139

3249


4

3

2

1

0

minus1

minus2

minus3

4

3

2

1

0

minus1

minus2

minus3

Stan

dard

ised

norm

alise

d vo

lum

Stan

dard

ised

norm

alise

d vo

lum







(A)

(A998400)

(a)

(A)

(A998400)

Tf A1AT

(b)

b1 b2

b3 b4

Patient Control

Tf

A1AT

(A) (B)

(c)



Table2MALD

ITOFTO

FMSspectro

metry

proteinidentifi

catio

nin

2DEmap

ofserum

Proteinname

Accessionnu

mber

Expressio

nlevellowast

Biologicalfunctio


Power

Fold

Max

CVdagger

MASC

OT

proteinscore

Sequ

ence

coverage

()

PIDagger

Mw(D

a)para

Keratin

1gi|7331218


000

079

004

099

2298

121

4816

66149

Keratin

10gi|18

6629

uarr000

079

004

099

2298

1145

472

39832

Cytokeratin

9gi|435476

uarr000

444

009

091

152634

174

5519

62320

Keratin

1gi|7331218

uarr000

444

009

091

152634

785

16816

66149

Serum

albu

min

gi|28592

uarr

Molecular

carriera

ndregu

latoro

fosm

otic

pressure

inbloo

d000

678

011

086

193642

264

6605

71316

Serum

albu

min

gi|28592

uarr000519

012

089

21

3832

149

4605

71316

Serum

albu

min

gi|28592

uarr000284

007

094

182876

308

9605

71316

Serum

albu

min

gi|28592

uarr000740

011

085

183143

632

605

71316

Haptoglob

inhp

2gi|223976

uarr

Free

hemoglobinbind

ing

proteinin

plasma(

iron

oxidativea

ctivity

supp

ressor)

000105

004

098

28

4480

115

6623

42344

Haptoglob

inhp

2gi|223976

uarr000372

008

092

23

4011

703

623

42344

Haptoglob

inhp

2gi|223976

uarr000705

011

086

32

4662

139

6623

42344

Haptoglob

inhp

2gi|223976

uarr000231

006

095

35

7920

279

11623

42344

Igkapp

agi|10

6529

uarrIm

mun

oglobu

lin000511

010

089

183777

5416

561

11161

Transfe

rrin

gi|339469

darrIron

transport

000

979

013

082

152225

503

840

80768

alph

a1antitrypsin

gi|17

7831

darrprotease

inhibitor

000159

005

097

192626

584

491

57706

Max

CVischosen

bycomparis

onso

fCVam

ongcontroland

patie

ntgrou

pslowastexpressio

nlevelsho

wsp

rotein

changesinthep

atient

incomparis

onwith

controlsdaggerMaxim

umof

coeffi

ciento

fvarianceo

btained

incomparis

onbetweencontroland

patie

ntgrou

pDaggerIsoelectric

point

para Molecular

weightinDalton








5 Conclusions


Abbreviations




Ethical Approval








Acknowledgment


References




































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


+ minus

(a)

+ minus

80768kD

54953kD

13800 kD

(b)


03

02

01

00

minus01




Prin

cipa

l com

pone

nts2

(a)

10090807060504030201001 2 3 4 5 6 7 8 9 10 11


Power analysis

Spot

s with

pow

ergt08

()

(b)





4 Discussion







Dendrogram

Dist

ance

20

15

10

05

00

1063

1313

1330

1331

1344

1377

1458

1466

1469

1481

1482

1490

1487

1524

1495

1558

1625

1662

2168

2487

2563

2570

2758

2796

2828

2837

2847

2888

2914

2921

3026

3107

3120

3139

3249


4

3

2

1

0

minus1

minus2

minus3

4

3

2

1

0

minus1

minus2

minus3

Stan

dard

ised

norm

alise

d vo

lum

Stan

dard

ised

norm

alise

d vo

lum







(A)

(A998400)

(a)

(A)

(A998400)

Tf A1AT

(b)

b1 b2

b3 b4

Patient Control

Tf

A1AT

(A) (B)

(c)



Table2MALD

ITOFTO

FMSspectro

metry

proteinidentifi

catio

nin

2DEmap

ofserum

Proteinname

Accessionnu

mber

Expressio

nlevellowast

Biologicalfunctio


Power

Fold

Max

CVdagger

MASC

OT

proteinscore

Sequ

ence

coverage

()

PIDagger

Mw(D

a)para

Keratin

1gi|7331218


000

079

004

099

2298

121

4816

66149

Keratin

10gi|18

6629

uarr000

079

004

099

2298

1145

472

39832

Cytokeratin

9gi|435476

uarr000

444

009

091

152634

174

5519

62320

Keratin

1gi|7331218

uarr000

444

009

091

152634

785

16816

66149

Serum

albu

min

gi|28592

uarr

Molecular

carriera

ndregu

latoro

fosm

otic

pressure

inbloo

d000

678

011

086

193642

264

6605

71316

Serum

albu

min

gi|28592

uarr000519

012

089

21

3832

149

4605

71316

Serum

albu

min

gi|28592

uarr000284

007

094

182876

308

9605

71316

Serum

albu

min

gi|28592

uarr000740

011

085

183143

632

605

71316

Haptoglob

inhp

2gi|223976

uarr

Free

hemoglobinbind

ing

proteinin

plasma(

iron

oxidativea

ctivity

supp

ressor)

000105

004

098

28

4480

115

6623

42344

Haptoglob

inhp

2gi|223976

uarr000372

008

092

23

4011

703

623

42344

Haptoglob

inhp

2gi|223976

uarr000705

011

086

32

4662

139

6623

42344

Haptoglob

inhp

2gi|223976

uarr000231

006

095

35

7920

279

11623

42344

Igkapp

agi|10

6529

uarrIm

mun

oglobu

lin000511

010

089

183777

5416

561

11161

Transfe

rrin

gi|339469

darrIron

transport

000

979

013

082

152225

503

840

80768

alph

a1antitrypsin

gi|17

7831

darrprotease

inhibitor

000159

005

097

192626

584

491

57706

Max

CVischosen

bycomparis

onso

fCVam

ongcontroland

patie

ntgrou

pslowastexpressio

nlevelsho

wsp

rotein

changesinthep

atient

incomparis

onwith

controlsdaggerMaxim

umof

coeffi

ciento

fvarianceo

btained

incomparis

onbetweencontroland

patie

ntgrou

pDaggerIsoelectric

point

para Molecular

weightinDalton








5 Conclusions


Abbreviations




Ethical Approval








Acknowledgment


References




































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Dendrogram

Dist

ance

20

15

10

05

00

1063

1313

1330

1331

1344

1377

1458

1466

1469

1481

1482

1490

1487

1524

1495

1558

1625

1662

2168

2487

2563

2570

2758

2796

2828

2837

2847

2888

2914

2921

3026

3107

3120

3139

3249


4

3

2

1

0

minus1

minus2

minus3

4

3

2

1

0

minus1

minus2

minus3

Stan

dard

ised

norm

alise

d vo

lum

Stan

dard

ised

norm

alise

d vo

lum







(A)

(A998400)

(a)

(A)

(A998400)

Tf A1AT

(b)

b1 b2

b3 b4

Patient Control

Tf

A1AT

(A) (B)

(c)



Table2MALD

ITOFTO

FMSspectro

metry

proteinidentifi

catio

nin

2DEmap

ofserum

Proteinname

Accessionnu

mber

Expressio

nlevellowast

Biologicalfunctio


Power

Fold

Max

CVdagger

MASC

OT

proteinscore

Sequ

ence

coverage

()

PIDagger

Mw(D

a)para

Keratin

1gi|7331218


000

079

004

099

2298

121

4816

66149

Keratin

10gi|18

6629

uarr000

079

004

099

2298

1145

472

39832

Cytokeratin

9gi|435476

uarr000

444

009

091

152634

174

5519

62320

Keratin

1gi|7331218

uarr000

444

009

091

152634

785

16816

66149

Serum

albu

min

gi|28592

uarr

Molecular

carriera

ndregu

latoro

fosm

otic

pressure

inbloo

d000

678

011

086

193642

264

6605

71316

Serum

albu

min

gi|28592

uarr000519

012

089

21

3832

149

4605

71316

Serum

albu

min

gi|28592

uarr000284

007

094

182876

308

9605

71316

Serum

albu

min

gi|28592

uarr000740

011

085

183143

632

605

71316

Haptoglob

inhp

2gi|223976

uarr

Free

hemoglobinbind

ing

proteinin

plasma(

iron

oxidativea

ctivity

supp

ressor)

000105

004

098

28

4480

115

6623

42344

Haptoglob

inhp

2gi|223976

uarr000372

008

092

23

4011

703

623

42344

Haptoglob

inhp

2gi|223976

uarr000705

011

086

32

4662

139

6623

42344

Haptoglob

inhp

2gi|223976

uarr000231

006

095

35

7920

279

11623

42344

Igkapp

agi|10

6529

uarrIm

mun

oglobu

lin000511

010

089

183777

5416

561

11161

Transfe

rrin

gi|339469

darrIron

transport

000

979

013

082

152225

503

840

80768

alph

a1antitrypsin

gi|17

7831

darrprotease

inhibitor

000159

005

097

192626

584

491

57706

Max

CVischosen

bycomparis

onso

fCVam

ongcontroland

patie

ntgrou

pslowastexpressio

nlevelsho

wsp

rotein

changesinthep

atient

incomparis

onwith

controlsdaggerMaxim

umof

coeffi

ciento

fvarianceo

btained

incomparis

onbetweencontroland

patie

ntgrou

pDaggerIsoelectric

point

para Molecular

weightinDalton








5 Conclusions


Abbreviations




Ethical Approval








Acknowledgment


References




































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(A)

(A998400)

(a)

(A)

(A998400)

Tf A1AT

(b)

b1 b2

b3 b4

Patient Control

Tf

A1AT

(A) (B)

(c)



Table2MALD

ITOFTO

FMSspectro

metry

proteinidentifi

catio

nin

2DEmap

ofserum

Proteinname

Accessionnu

mber

Expressio

nlevellowast

Biologicalfunctio


Power

Fold

Max

CVdagger

MASC

OT

proteinscore

Sequ

ence

coverage

()

PIDagger

Mw(D

a)para

Keratin

1gi|7331218


000

079

004

099

2298

121

4816

66149

Keratin

10gi|18

6629

uarr000

079

004

099

2298

1145

472

39832

Cytokeratin

9gi|435476

uarr000

444

009

091

152634

174

5519

62320

Keratin

1gi|7331218

uarr000

444

009

091

152634

785

16816

66149

Serum

albu

min

gi|28592

uarr

Molecular

carriera

ndregu

latoro

fosm

otic

pressure

inbloo

d000

678

011

086

193642

264

6605

71316

Serum

albu

min

gi|28592

uarr000519

012

089

21

3832

149

4605

71316

Serum

albu

min

gi|28592

uarr000284

007

094

182876

308

9605

71316

Serum

albu

min

gi|28592

uarr000740

011

085

183143

632

605

71316

Haptoglob

inhp

2gi|223976

uarr

Free

hemoglobinbind

ing

proteinin

plasma(

iron

oxidativea

ctivity

supp

ressor)

000105

004

098

28

4480

115

6623

42344

Haptoglob

inhp

2gi|223976

uarr000372

008

092

23

4011

703

623

42344

Haptoglob

inhp

2gi|223976

uarr000705

011

086

32

4662

139

6623

42344

Haptoglob

inhp

2gi|223976

uarr000231

006

095

35

7920

279

11623

42344

Igkapp

agi|10

6529

uarrIm

mun

oglobu

lin000511

010

089

183777

5416

561

11161

Transfe

rrin

gi|339469

darrIron

transport

000

979

013

082

152225

503

840

80768

alph

a1antitrypsin

gi|17

7831

darrprotease

inhibitor

000159

005

097

192626

584

491

57706

Max

CVischosen

bycomparis

onso

fCVam

ongcontroland

patie

ntgrou

pslowastexpressio

nlevelsho

wsp

rotein

changesinthep

atient

incomparis

onwith

controlsdaggerMaxim

umof

coeffi

ciento

fvarianceo

btained

incomparis

onbetweencontroland

patie

ntgrou

pDaggerIsoelectric

point

para Molecular

weightinDalton








5 Conclusions


Abbreviations




Ethical Approval








Acknowledgment


References




































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table2MALD

ITOFTO

FMSspectro

metry

proteinidentifi

catio

nin

2DEmap

ofserum

Proteinname

Accessionnu

mber

Expressio

nlevellowast

Biologicalfunctio


Power

Fold

Max

CVdagger

MASC

OT

proteinscore

Sequ

ence

coverage

()

PIDagger

Mw(D

a)para

Keratin

1gi|7331218


000

079

004

099

2298

121

4816

66149

Keratin

10gi|18

6629

uarr000

079

004

099

2298

1145

472

39832

Cytokeratin

9gi|435476

uarr000

444

009

091

152634

174

5519

62320

Keratin

1gi|7331218

uarr000

444

009

091

152634

785

16816

66149

Serum

albu

min

gi|28592

uarr

Molecular

carriera

ndregu

latoro

fosm

otic

pressure

inbloo

d000

678

011

086

193642

264

6605

71316

Serum

albu

min

gi|28592

uarr000519

012

089

21

3832

149

4605

71316

Serum

albu

min

gi|28592

uarr000284

007

094

182876

308

9605

71316

Serum

albu

min

gi|28592

uarr000740

011

085

183143

632

605

71316

Haptoglob

inhp

2gi|223976

uarr

Free

hemoglobinbind

ing

proteinin

plasma(

iron

oxidativea

ctivity

supp

ressor)

000105

004

098

28

4480

115

6623

42344

Haptoglob

inhp

2gi|223976

uarr000372

008

092

23

4011

703

623

42344

Haptoglob

inhp

2gi|223976

uarr000705

011

086

32

4662

139

6623

42344

Haptoglob

inhp

2gi|223976

uarr000231

006

095

35

7920

279

11623

42344

Igkapp

agi|10

6529

uarrIm

mun

oglobu

lin000511

010

089

183777

5416

561

11161

Transfe

rrin

gi|339469

darrIron

transport

000

979

013

082

152225

503

840

80768

alph

a1antitrypsin

gi|17

7831

darrprotease

inhibitor

000159

005

097

192626

584

491

57706

Max

CVischosen

bycomparis

onso

fCVam

ongcontroland

patie

ntgrou

pslowastexpressio

nlevelsho

wsp

rotein

changesinthep

atient

incomparis

onwith

controlsdaggerMaxim

umof

coeffi

ciento

fvarianceo

btained

incomparis

onbetweencontroland

patie

ntgrou

pDaggerIsoelectric

point

para Molecular

weightinDalton








5 Conclusions


Abbreviations




Ethical Approval








Acknowledgment


References




































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








5 Conclusions


Abbreviations




Ethical Approval








Acknowledgment


References




































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Acknowledgment


References




































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Comparative Proteomic Study Reveals the ...

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