Research Article Differential Proteomics Analysis …downloads.hindawi.com/journals/bmri/2016/4814702.pdfResearch Article Differential Proteomics Analysis of Colonic Tissues in Patients

Research ArticleDifferential Proteomics Analysis of Colonic Tissues inPatients of Slow Transit Constipation

Songlin Wan Weicheng Liu Cuiping Tian Xianghai Ren Zhao Ding Qun QianCongqing Jiang and Yunhua Wu

Zhongnan Hospital of Wuhan University Department of Colorectal amp Anal Surgery Clinical Center of Intestinal andColorectal Diseases of Hubei Province Key Laboratory of Intestinal amp Colorectal Diseases of Hubei Province Wuhan UniversityNo 169 Donghu Road Wuchang District Wuhan Hubei 430071 China

Correspondence should be addressed to Congqing Jiang wb002554whueducn and Yunhua Wu wb002564whueducn

Received 2 March 2016 Accepted 11 April 2016

Academic Editor Terry K Smith

Copyright copy 2016 Songlin Wan et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objective To investigate and screen the different expression of proteins in STC and normal group with a comparative proteomicapproachMethods Two-dimensional electrophoresis was applied to separate the proteins in specimens from both 5 STC patientsand 5 normal controls The proteins with statistically significant differential expression between two groups were identified bycomputer aided image analysis and matrix assisted laser desorption ionization tandem time of flight mass spectrometry (MALDI-TOF-MS) Results A total of 239 protein spots were identified in the average gel of the normal control and 215 in patients with STCA total of 197 protein spots were matched and the mean matching rate was 82There were 14 protein spots which were expressedwith statistically significant differences from others Of those 14 protein spots the expression of 12 spots increased markedly whilethat of 2 spots decreased significantly Conclusion The proteomics expression in colonic specimens of STC patients is statisticallysignificantly different from that of normal control which may be associated with the pathogenesis of STC

1 Introduction

Constipation is a very common functional gastrointestinaldisorder that affects patientsrsquo quality of life [1 2] The preva-lence of constipation in the worldwide general population isvery variable ranging from 2 to 35 in adults and rangingfrom 07 to 296 in children [3] Slow transit constipation(STC) is common type of chronic constipation which affects13ndash37 of patients who have chronic treatment-resistantconstipation [4 5] The epidemiological data show that thereis a higher incidence of STC in young females than in males[6 7]

STC is characterized by slow proximal colonic transit andprolongation of transit time through the colon which canbe demonstrated with radiopaque marker transit tests [8 9]Despite its high prevalence its etiology and precise mech-anism(s) remain unknown [4 10] and STC have becomethe main subject of many colorectal and anal surgeriesAlthough several morphological changes have been reportedthe exact mechanism(s) of STC pathogenesis remains poorly

understood and there are no effective clinical treatmentsEven if some patientsrsquo clinical symptoms may improve afterconservative management the small subset of STC patientswho do not respond to conservative management are consid-ered candidates for surgery [11]

At present quantitative proteomic analysis has becomean important approach to screen the key protein of thepathogenesis of diseases ldquoProteomerdquo first referred to thetotal protein complement encoded by a given genome Nowit comprises any isoforms posttranslational modificationsinteractions and actually everything ldquopostgenomicrdquo [12 13]The research about proteomics involves large scale detectionidentification and characterization of proteins which ishighly promising for biomarker detection overmany diseases[14 15] The most common method applied is a combinationof two-dimensional electrophoresis (2DE) and mass spec-trometry

In our study we separate protein in test group and con-trol group through two-dimensional electrophoresis (2DE)ImageMaster 2D Elite computer aided image analysis and

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 4814702 6 pageshttpdxdoiorg10115520164814702

2 BioMed Research International

matrix assisted laser desorption ionization tandem time offlight mass spectrometry (MALDI-TOF-MS) to find out thekey protein acting in the pathogenesis of STC

2 Materials and Methods

21 General Information Patients were divided into a testgroup and a control group each group included 5 cases Allpatients had undergone a rigorous selection First all candi-dates should have prior screening through repeated gastroin-testinal transit time (GITT) study using 20 radiopaquemark-ers and abdominal imaging tests performed on 6 h days 1 2and 3 And the abdominal imaging test performed on 6h aftertaking 20 radiopaque markers was used to identify whetherthere was slow transit in the terminal intestine All these fiveSTC patients showed slow transit mainly on the left colonthrough GITT study and all samples were collected from theleft hemicolon and the same regions were available for bothSTC and control groups Second autoimmune diseases suchas diabetes mellitus type I autoimmune enteric leiomyositisrheumatoid arthritis systemic sclerosis and systemic lupuserythematosus can prolong colonic transit time by damageof colonic smooth muscle cells And all candidates with anyof these autoimmune diseases were excluded and all thesefive STC patients selected were without any autoimmunediseases at all Other evaluations including colonoscopybarium enema defecography and anorectalmanometrywereused to rule out colorectal neoplasms megacolon outletobstruction and pelvic floor dysfunction The conventionalmedical therapy had failed in all STC patients Conservativetreatment did not improve their bowel habits and defecationfrequently had to be achieved by the application of stimuluslaxatives and enemas All STC patients (119899 = 5 mean age 50years range 43ndash81 years 5 women and 0 men) underwentsubtotal colectomy with antiperistaltic cecoproctostomy ortotal colectomy with ileorectal anastomosis Patients in thecontrol group (119899 = 5 mean age 52 years range 41ndash79 years 5women and 0 men) underwent partial colectomy for incom-plete intestinal obstruction caused by colorectal neoplasmsand the control tissue was taken from the proximal endAll control patients were reported with normal bowel habitsAll specimens were obtained immediately after resection andsnap-frozen in liquid nitrogen until being used

The high-abundance proteins were removed with thesteps recommended by Calbiochem Company and theimproved Bradford method was used for sample proteinquantitation [16]

22 Two-Dimensional Electrophoresis (2D)

221 Extraction and Determination of the Concentrationof Protein A 20mL sterile EP tube was prepared About200mg small piece of tissue was rapidly clipped from thespecimen andweighed on electronic scale and then the tissuewas placed in the precooling Petri dish with normal saline(6mL 09 NaCl) The tissue was shredded in the dishand then transferred to 15mL EP tube 800120583L cold proteinlysate (containing a protease inhibitor) was added into theEP tube which was to be quickly transferred to an ultrasonic

homogenizerThe EP tubewas coatedwith iceThe ultrasonichomogenizer was triggered 5 times 3 seconds each timeThetube was statically placed for 30 minutes the supernatantwas collected by centrifugation (4000 rmin 4∘C 60min) andthen stored in the minus80∘C refrigerator

222 Protein Purification The protein sample was trans-ferred to a labeled EP tube (2mL) After adding proteincracking fluid with 3 times volume of samples the mixturewas shock-cracked and subsequently placed on ice for 15minThen themixturewas centrifuged for 8minwith a high speedof 12000 rmin at 10∘C The supernatant was discarded andthe precipitation was resuspended and was shock-cracked for30 s every 10min for 3 cycles Finally the precipitation wasresuspended again and used for next steps

223 Concentration Determination Protein concentrationswere determined by the instruction of the Bio-Rad RCDCkit manual The specific steps were as follows standardconcentration and gradient BSA reagent were prepared andthe concentrations were respectively 0 025 05 075 1 125and 15 (gmL) Reagent A working fluid resulted from themix of 20120583L DC Reagent S and 1mL DC Reagent A Eight15mL EP tubes were respectively added with 25 120583L standardBCA solution and 5-fold dilution of the sample and thenin each tube 125 120583L RC Reagent I and RC Reagent II wereaddedThemix was collected by centrifugation (15000 rmin5min) after the supernatant was discarded 127120583L ReagentA was added to the tubes The tubes were statically placedfor 5 minutes after blending Then 1mL DC Reagent B wasadded to each tube and blended Absorbance values (750 nm)weremeasured after statically placed at room temperature for15min

224 Isoelectrofocusing (IEF) Rehydration stock solutionwith IPG buffer was dissolved at room temperature (DTTand Bio-Lyte were added before using) The proper amountof rehydration stock solution with IPG buffer was addedto samples till a final volume of 480 120583L according to theconcentration of the samples The sample was linearly addedalong with the extension of the focusing plate The protectivelayer on the surface of the gel was stripped with tweezersso as to distinguish positive and negative electrodes andthen the gel was placed downside onto the sample solutionin the focusing plate 2mL mineral oil was covered by eachgel The mineral oil was slowly added onto the support filmdrop by drop IEF was carried out at 50V (14 h) 250V linear(30min) 1000V fast (60min) 10000V linear (1 h) 10000Vfast 80000V and 500V (arbitrary time)

225 SDS-PAGE Electrophoresis Two 10 acrylamide gelswere prepared 1 cm was reserved on the upper side and cov-ered with water-saturated n-butanol and then polymerizedfor 30min The n-butanol was then discarded The gels werewashed three times with ultrapure water Water on the glassplate was sucked with filter paper The gels were taken to thehydration plates and 6mL equilibrium liquid I (02 g DTTper 10mL equilibrium mother liquid before using) and II

BioMed Research International 3

(a) (b)

Figure 1 Comparison of two-dimensional (2D) gel electrophoresis results using colonic tissues between STC and control groups STC group(a) and control group (b) 2D gel electrophoresis

(025 g iodoacetamide per 10mL equilibrium mother liquidbefore using) were then added into each plate The platewas placed on a shaker (15min) The strip was carefullyplaced on the surface of the gel Air bubble between themwas removed The heated sealant was cooled to 37∘C andthen applied to the surface of gel The gel was transferredto the electrophoresis tank after the sealant was solidifiedTheupper electrophoresis tankwas filledwith electrophoresissolution after checking the positive and negative chargesThe electrophoresis was first carried out at Low Voltage(5mAgel) within 2 cm downside the sample and then theelectrophoresis was carried out to the bottom of the samplewith a current of 30mAgel

226 Coomassie Blue Staining Gels were dyed with Coo-massie Blue for 16 h after being fixed with 12 TCA for 2 hThen the gels were washed with 01molL Tris-H

3PO4(pH

65) for 2min and then with 25 alcohol for 1min at most Atlast the gels were fixed with protein-dye compounds again

227 Gel analysis After scanned by Image Scanner II trans-mission scanner and marked with Scan scanning softwarethe gels were analyzed by image analysis software (ImageMas-ter 2D Elite50) And plots with expression differences morethan 10 times (119875 lt 005) were chosen as targeted proteins

23 Mass Spectrum Identification

231 Enzymatic Hydrolysis Targeted parts of the gels wereremoved and washed with ultrapure water and then decol-orized with 200120583Lmixed liquid (25mmolLNH

4HCO3 50

ACN) for 20min at 37∘C An alternative procedure wasultrasonic decoloring Then 100 120583L ACN was added in themixture and discarded when the color of the gels turns whiteSubsequently the gels were enzymatically hydrolyzed withTrypsin (diluted with 25mmolLNH

4HCO3at a concentra-

tion of 125mgmL) for 45min at 4∘C Finally gels werecontinuously hydrolyzed with 25mmolLNH

4HCO310 120583L at

37∘C for 16 h

232 Mass Spectrometry Analysis Substrate (4mg HCCA in1mL solution of (ACN 01TFA (70 30))) was prepared theenzymatically hydrolyzed gels were dried and covered with01 120583L substrate Then the mixture was diluted 25 times itsoriginal volume and salts were removed using 01 TFA

233 Database Analysis Data frommass spectrometry wereanalyzed and screened with Swiss-Prot Database in Mascot

24 Statistical Analysis Statistical analyses were performedusing SPSS 190 software for Windows (SPSS Chicago ILUSA) All groups of variables were tested for normal dis-tribution using the Kolmogorov-Smirnov test and normallydistributed data were expressed as mean plusmn SD followed by atwo-tailed Studentrsquos 119905 test to determine 119875 values All resultswith 119875 lt 005 were considered statistically significant

All research involving human participants was approvedby the Zhongnan Hospital of Wuhan University EthicsCommittee and we obtained written informed consent fromall participants before they were enrolled in the study (ethicalconsiderations ethics number 2010010 ethical approvalstarting date March 8 2010 ethical approval expiration dateMarch 8 2013)

3 Results

(1) Two-dimensional electrophoresis in proteomics researchfind that there were 14 differential protein spots between thetwo groups and the expression difference were more than 10times (Figure 1)

(2) Mass spectrum (MS) identification and databasesearch after the protein spots with more than 10-fold dif-ference between two groups were removed for the gels theywere subsequently dealt with using the following proceduresincluding enzyme digestion extraction of the peptides andsample desalination Finally peptide mass fingerprint (PMF)and MSMS spectrum were achieved through MALDI-TOF-MS analysis of proteins

The retrieval for mass spectrometric data in Swiss-Prot successfully identified anterior 14 spots wherein the


Table 1 Elevated expression proteins in colonic tissues of STCpatients compared with control group

Name Score pI Formula weight Fold changeACTG 82 531 42108 1791ENPL 80 476 92696 2930F102B 80 662 39911 1190GBB1 76 560 38151 1598GELS 80 590 86043 1372K1C9 63 514 62255 2001MTMR7 62 594 76754 1592PDIA1 91 476 57480 2271PDIA6 68 495 48490 3079PFD6 78 883 14574 1021PRVA 60 498 12051 1531TRFE 79 681 79294 1731

Table 2 Decreased expression proteins in colonic tissues of STCpatients compared with control group

Name Score pI Formula weight Fold changeMRP4 99 841 150344 minus3286VIME 81 506 53676 minus1239

expression of 12 spots is up and that of 2 spots is downin STC group (Tables 1 and 2) Our further studies showthat multidrug resistance-associated protein 4 (MRP4) is ofclinical significance (Figure 2) MRP4 is playing a critical rolein the process of maintaining the stability of smooth musclecells (SMC) [16 17]The downregulation ofMRP4 expressionin colon tissue of STC patients may be related to the decreaseof SMC and may play a role in the pathogenesis of STC byaffecting colon transmission

4 Discussion

STC pathogenesis and mechanism not yet completelyexpounded that at present clinical STC patients mainlydepend on purgative and gastrointestinal prokinetic agentsfor conservative management A few clinical studies haveconfirmed that pharmacotherapy is effective in treating STCbut most patients with STC to accept drug treatment do notquite approve of the effect The effectiveness of current STCdrugs is fairly limited The abuse of laxatives to assist defe-cation of patients with STC in clinical is very common somepatients who do not respond to conservativemanagement areconsidered candidates for surgery [11]

Proteomics has been used to study the specific proteingroup that is functioning on different space and time andthe study included three dimensions expression proteomicsfunctional proteomics and structural proteomics Throughthe former three dimensions of study researchers explore thefunctionmechanism regulatory mechanism and interactionmechanism within group of corresponding proteins at thelevel of protein which provide a theoretical reference forclinical diagnosis pathologic study drug screening andpathogenesis of disease STC currently facesmany challenges

1638846

1066599 1449735

842518

13086621209631

1707765

1916917951485

1542755

25011581799878

1164593

230308721029552584110

3312159

0

250

500

750

1000

1250

Inte

nsity

(au

)

1500 2000 2500 30001000mz

(a)

605040

0

5

10

Num

ber o

f hits

(b)

Figure 2 Mass spectrum identification figure of protein MRP4 andits matched analysis diagramMass spectrum identification figure ofprotein MRP4 (a) and its matched analysis diagram (b)

ranging from the elucidation of its pathophysiology to theeffective treatment in patients Proteomics has been widelyemployed in many diseases in the search of biomarkersparticularly cancer proteins It has great potential to improveboth our understanding and clinical management of STC Inthe field of surgical research proteomics studies mainly focuson tumor-related diseases at present the proteomics studiesfor functional diseases are quite few and the proteomicsstudy for STC is not reported so far In our proteomicsstudies we find that compared with control group theexpression of 12 protein spots increased markedly and that of2 protein spots decreased significantly in STC group Amongthe low expression of proteins of STC multidrug resistance-associated protein 4 (MRP4) is of clinical significance

MRP4 belongs to transmembrane protein family itsmain function is to transport intracellular cyclic nucleotidesthat include cyclic adenosine monophosphate and cyclicguanosine monophosphate Some studies have confirmedthat MRP4 participated in cellular excretion of resveratrol3-O-glucuronide and resveratrol 41015840-O-glucuronide [18] theinhibition of urinary excretion of methotrexate [19] cellularexcretion of the raloxifene sulfates in breast cancer patients[20] the process of human obstructive cholestasis [21] and soon Recently studies have revealed thatMRP4 can be involvedin maintaining the homeostasis of smoothmuscle cell (SMC)by the regulation of intracellular cyclic nucleotide levels onthe intracellular cyclic nucleotide signaling pathways (PKC


PKA etc)When the level of intracellularMRP4 is decreasingor inhibited the significant reduction of the stability of SMCresults in the decrease of proliferation cells and increaseof apoptosis which may be involved in the pathogenesisof idiopathic pulmonary arterial hypertension (IPAH) andcoronary heart disease (CHD) [16 17] Our previous studiesabout STC have shown that compared with normal colonictissue the number of SMC decreases and the apoptosisof SMC increases in colonic tissue with STC which maybe involved in the pathogenesis of STC In this study thehomeostasis of SMC in colonic specimens of STC patients ischanged and the decrease of proliferation cells and increase ofapoptosis may result from the reduction ofMRP4 expressionwhich may play a role in the pathogenesis of STC

The results suggest that the different expression of pro-teins between STC and normal group may be associatedwith the pathogenesis of STC The pathogenesis mechanismof STC remains largely obscure which may result frommultifactor the change of the expression of MRP4 may playa certain role Further studies for the relationship betweenMRP4 and the pathogenesis mechanism of STC and targeteddrugs to the change of MRP4 are required

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Songlin Wan and Weicheng Liu contributed equally to thiswork

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (nos 81500505 and 81570492) theYoung and Middle-Aged Medical Talent Support Programof the Health and Family Planning Commission of WuhanMunicipality the Natural Science Foundation of HubeiProvince of China (nos 2015CFB636 and 2011CDB521)Central Universities and Scientific Research Foundation ofWuhan University (no 303410500160) the Natural ScienceFoundation of Zhongnan Hospital of Wuhan University (no22)

References

[1] D Polymeros I Beintaris A Gaglia et al ldquoPartially hydrolyzedguar gum accelerates colonic transit time and improves symp-toms in adultswith chronic constipationrdquoDigestiveDiseases andSciences vol 59 no 9 pp 2207ndash2214 2014

[2] L Leung T Riutta J Kotecha and W Rosser ldquoChronic con-stipation an evidence-based reviewrdquo Journal of the AmericanBoard of Family Medicine vol 24 no 4 pp 436ndash451 2011

[3] S M Mugie M A Benninga and C Di Lorenzo ldquoEpidemiol-ogy of constipation in children and adults a systematic reviewrdquoBest Practice and Research Clinical Gastroenterology vol 25 no1 pp 3ndash18 2011

[4] A Lembo andM Camilleri ldquoCurrent concepts chronic consti-pationrdquo The New England Journal of Medicine vol 349 no 14pp 1360ndash1368 2003

[5] S Singh S Heady E Coss-Adame and S S C Rao ldquoClinicalutility of colonic manometry in slow transit constipationrdquoNeurogastroenterology and Motility vol 25 no 6 pp 487ndash4952013

[6] C H Knowles S M Scott C Rayner et al ldquoIdiopathic slow-transit constipation an almost exclusively female disorderrdquoDiseases of the Colon and Rectum vol 46 no 12 pp 1716ndash17172003

[7] D M Preston and J E Lennard-Jones ldquoSevere chronic consti-pation of young women lsquoidiopathic slow transit constipationrsquordquoGut vol 27 no 1 pp 41ndash48 1986

[8] M Camilleri W Grant Thompson J W Fleshman and J HPemberton ldquoClinical management of intractable constipationrdquoAnnals of Internal Medicine vol 121 no 7 pp 520ndash528 1994

[9] M Bouchoucha G Devroede P Arhan et al ldquoWhat is themeaning of colorectal transit time measurementrdquo Diseases ofthe Colon amp Rectum vol 35 no 8 pp 773ndash782 1992

[10] A Emmanuel M Cools L Vandeplassche and R KerstensldquoPrucalopride improves bowel function and colonic transit timein patients with chronic constipation an integrated analysisrdquoThe American Journal of Gastroenterology vol 109 no 6 pp887ndash894 2014

[11] M A Levitt K L Mathis and J H Pemberton ldquoSurgicaltreatment for constipation in children and adultsrdquo Best Practiceand Research Clinical Gastroenterology vol 25 no 1 pp 167ndash179 2011

[12] V C Wasinger S J Cordwell A Cerpa-Poljak et al ldquoProgresswith gene-product mapping of the Mollicutes Mycoplasmagenitaliumrdquo Electrophoresis vol 16 no 7 pp 1090ndash1094 1995

[13] A J Ferreri G Illerhaus E Zucca F Cavalli and InternationalExtranodal Lymphoma Study Group ldquoFlows and flaws inprimary central nervous system lymphomardquo Nature ReviewsClinical Oncology vol 7 no 8 2010

[14] A Vaiopoulou M Gazouli G Theodoropoulos and GZografos ldquoCurrent advantages in the application of proteomicsin inflammatory bowel diseaserdquoDigestive Diseases and Sciencesvol 57 no 11 pp 2755ndash2764 2012

[15] P P Chan V C Wasinger and R W Leong ldquoCurrent appli-cation of proteomics in biomarker discovery for inflammatorybowel diseaserdquo World Journal of Gastrointestinal Pathophysiol-ogy vol 7 no 1 pp 27ndash37 2016

[16] Y Hara Y Sassi C Guibert et al ldquoInhibition ofMRP4 preventsand reverses pulmonary hypertension in micerdquo The Journal ofClinical Investigation vol 121 no 7 pp 2888ndash2897 2011

[17] Y Sassi L Lipskaia G Vandecasteele et al ldquoMultidrugresistance-associated protein 4 regulates cAMP-dependent sig-naling pathways and controls human and rat SMC prolifera-tionrdquo The Journal of Clinical Investigation vol 118 no 8 pp2747ndash2757 2008

[18] S Wang F Li E Quan D Dong and B Wu ldquoEffluxtransport characterization of resveratrol glucuronides in UDP-glucuronosyltransferase 1A1 transfected hela cells applicationof a cellular pharmacokinetic model to decipher the con-tribution of multidrug resistance-associated protein 4rdquo DrugMetabolism and Disposition vol 44 no 4 pp 485ndash488 2016

[19] A Kawase T Yamamoto S Egashira and M Iwaki ldquoStereose-lective inhibition of methotrexate excretion by glucuronides ofnonsteroidal anti-inflammatory drugs via multidrug resistance


proteins 2 and 4rdquoThe Journal of Pharmacology and Experimen-tal Therapeutics vol 356 no 2 pp 366ndash374 2016

[20] X Zhou SWang H Sun and BWu ldquoSulfonation of raloxifenein HEK293 cells overexpressing SULT1A3 involvement ofbreast cancer resistance protein (BCRPABCG2) andmultidrugresistance-associated protein 4 (MRP4ABCC4) in excretionof sulfate metabolitesrdquo Drug Metabolism and Pharmacokineticsvol 30 no 6 pp 425ndash433 2015

[21] W Lian X Liu L Yang et al ldquoThe role of TNFalpha inpromoting hepatic MRP4 expression via the p38-Rb-E2F1pathway in human obstructive cholestasisrdquo Biochemical andBiophysical Research Communications 2016

Submit your manuscripts athttpwwwhindawicom

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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


matrix assisted laser desorption ionization tandem time offlight mass spectrometry (MALDI-TOF-MS) to find out thekey protein acting in the pathogenesis of STC

2 Materials and Methods

21 General Information Patients were divided into a testgroup and a control group each group included 5 cases Allpatients had undergone a rigorous selection First all candi-dates should have prior screening through repeated gastroin-testinal transit time (GITT) study using 20 radiopaquemark-ers and abdominal imaging tests performed on 6 h days 1 2and 3 And the abdominal imaging test performed on 6h aftertaking 20 radiopaque markers was used to identify whetherthere was slow transit in the terminal intestine All these fiveSTC patients showed slow transit mainly on the left colonthrough GITT study and all samples were collected from theleft hemicolon and the same regions were available for bothSTC and control groups Second autoimmune diseases suchas diabetes mellitus type I autoimmune enteric leiomyositisrheumatoid arthritis systemic sclerosis and systemic lupuserythematosus can prolong colonic transit time by damageof colonic smooth muscle cells And all candidates with anyof these autoimmune diseases were excluded and all thesefive STC patients selected were without any autoimmunediseases at all Other evaluations including colonoscopybarium enema defecography and anorectalmanometrywereused to rule out colorectal neoplasms megacolon outletobstruction and pelvic floor dysfunction The conventionalmedical therapy had failed in all STC patients Conservativetreatment did not improve their bowel habits and defecationfrequently had to be achieved by the application of stimuluslaxatives and enemas All STC patients (119899 = 5 mean age 50years range 43ndash81 years 5 women and 0 men) underwentsubtotal colectomy with antiperistaltic cecoproctostomy ortotal colectomy with ileorectal anastomosis Patients in thecontrol group (119899 = 5 mean age 52 years range 41ndash79 years 5women and 0 men) underwent partial colectomy for incom-plete intestinal obstruction caused by colorectal neoplasmsand the control tissue was taken from the proximal endAll control patients were reported with normal bowel habitsAll specimens were obtained immediately after resection andsnap-frozen in liquid nitrogen until being used

The high-abundance proteins were removed with thesteps recommended by Calbiochem Company and theimproved Bradford method was used for sample proteinquantitation [16]

22 Two-Dimensional Electrophoresis (2D)

221 Extraction and Determination of the Concentrationof Protein A 20mL sterile EP tube was prepared About200mg small piece of tissue was rapidly clipped from thespecimen andweighed on electronic scale and then the tissuewas placed in the precooling Petri dish with normal saline(6mL 09 NaCl) The tissue was shredded in the dishand then transferred to 15mL EP tube 800120583L cold proteinlysate (containing a protease inhibitor) was added into theEP tube which was to be quickly transferred to an ultrasonic

homogenizerThe EP tubewas coatedwith iceThe ultrasonichomogenizer was triggered 5 times 3 seconds each timeThetube was statically placed for 30 minutes the supernatantwas collected by centrifugation (4000 rmin 4∘C 60min) andthen stored in the minus80∘C refrigerator

222 Protein Purification The protein sample was trans-ferred to a labeled EP tube (2mL) After adding proteincracking fluid with 3 times volume of samples the mixturewas shock-cracked and subsequently placed on ice for 15minThen themixturewas centrifuged for 8minwith a high speedof 12000 rmin at 10∘C The supernatant was discarded andthe precipitation was resuspended and was shock-cracked for30 s every 10min for 3 cycles Finally the precipitation wasresuspended again and used for next steps

223 Concentration Determination Protein concentrationswere determined by the instruction of the Bio-Rad RCDCkit manual The specific steps were as follows standardconcentration and gradient BSA reagent were prepared andthe concentrations were respectively 0 025 05 075 1 125and 15 (gmL) Reagent A working fluid resulted from themix of 20120583L DC Reagent S and 1mL DC Reagent A Eight15mL EP tubes were respectively added with 25 120583L standardBCA solution and 5-fold dilution of the sample and thenin each tube 125 120583L RC Reagent I and RC Reagent II wereaddedThemix was collected by centrifugation (15000 rmin5min) after the supernatant was discarded 127120583L ReagentA was added to the tubes The tubes were statically placedfor 5 minutes after blending Then 1mL DC Reagent B wasadded to each tube and blended Absorbance values (750 nm)weremeasured after statically placed at room temperature for15min

224 Isoelectrofocusing (IEF) Rehydration stock solutionwith IPG buffer was dissolved at room temperature (DTTand Bio-Lyte were added before using) The proper amountof rehydration stock solution with IPG buffer was addedto samples till a final volume of 480 120583L according to theconcentration of the samples The sample was linearly addedalong with the extension of the focusing plate The protectivelayer on the surface of the gel was stripped with tweezersso as to distinguish positive and negative electrodes andthen the gel was placed downside onto the sample solutionin the focusing plate 2mL mineral oil was covered by eachgel The mineral oil was slowly added onto the support filmdrop by drop IEF was carried out at 50V (14 h) 250V linear(30min) 1000V fast (60min) 10000V linear (1 h) 10000Vfast 80000V and 500V (arbitrary time)

225 SDS-PAGE Electrophoresis Two 10 acrylamide gelswere prepared 1 cm was reserved on the upper side and cov-ered with water-saturated n-butanol and then polymerizedfor 30min The n-butanol was then discarded The gels werewashed three times with ultrapure water Water on the glassplate was sucked with filter paper The gels were taken to thehydration plates and 6mL equilibrium liquid I (02 g DTTper 10mL equilibrium mother liquid before using) and II


(a) (b)




3PO4(pH





4HCO3 50




4HCO310 120583L at

37∘C for 16 h





3 Results










4 Discussion



1638846

1066599 1449735

842518

13086621209631

1707765

1916917951485

1542755

25011581799878

1164593

230308721029552584110

3312159

0

250

500

750

1000

1250

Inte

nsity

(au

)

1500 2000 2500 30001000mz

(a)

605040

0

5

10

Num

ber o

f hits

(b)







Competing Interests




Acknowledgments


References































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)




3PO4(pH





4HCO3 50




4HCO310 120583L at

37∘C for 16 h





3 Results










4 Discussion



1638846

1066599 1449735

842518

13086621209631

1707765

1916917951485

1542755

25011581799878

1164593

230308721029552584110

3312159

0

250

500

750

1000

1250

Inte

nsity

(au

)

1500 2000 2500 30001000mz

(a)

605040

0

5

10

Num

ber o

f hits

(b)







Competing Interests




Acknowledgments


References































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







4 Discussion



1638846

1066599 1449735

842518

13086621209631

1707765

1916917951485

1542755

25011581799878

1164593

230308721029552584110

3312159

0

250

500

750

1000

1250

Inte

nsity

(au

)

1500 2000 2500 30001000mz

(a)

605040

0

5

10

Num

ber o

f hits

(b)







Competing Interests




Acknowledgments


References































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Competing Interests




Acknowledgments


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








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Differential Proteomics Analysis …downloads.hindawi.com/journals/bmri/2016/4814702.pdfResearch Article Differential Proteomics Analysis of Colonic Tissues in Patients