Quantitative Proteomic Analysis Reveals the Deregulation of...

Research ArticleQuantitative Proteomic Analysis Revealsthe Deregulation of Nicotinamide Adenine DinucleotideMetabolism and CD38 in Inflammatory Bowel Disease

LongGui Ning1 Guodong Shan1 Zeyu Sun2 Fenming Zhang1 Chengfu Xu1

Xinhe Lou1 Sha Li1 Haojie Du1 Hongtan Chen1 and Guoqiang Xu 1

1Department of Gastroenterology First Affiliated Hospital Zhejiang University School of Medicine Hangzhou China2Proteomics amp Metabolomics Platform State Key Laboratory for Diagnosis and Treatment of Infectious DiseasesThe First Affiliated Hospital Hangzhou China

Correspondence should be addressed to Guoqiang Xu 1193065zjueducn

Received 27 December 2018 Revised 25 March 2019 Accepted 2 April 2019 Published 23 April 2019

Academic Editor David Bernardo

Copyright copy 2019 LongGui Ning 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

Inflammatory bowel disease (IBD) has become a major health challenge worldwide However the precise etiological andpathophysiological factors involved in IBD remain unclear Proteomics can be used for large-scale protein identification analysis Inthe current study using tandemmass tag- (TMT-) based shotgun proteomics proteomic differences between intestinal tissue fromhealth controls patients with Crohnrsquos disease (CD) and patients with ulcerative colitis (UC) were compared Proteins with foldchange gt2 or lt05 and P value lt 005 between groups were considered differentially expressed ProteinAtlas was used to analyze thetissue specificity of differentially expressed proteins (DEPs) Reactome pathway analysis was applied to cluster functional pathwaysA total of 4786 proteins were identified with 59 proteins showing higher levels and 43 showing lower levels in patients with IBDthan in controls Seventeen proteins including angiotensin converting enzyme 2 (ACE2) and angiotensin converting enzyme 1(ACE) showed higher levels in CD than in UC Several novel proteins such as CD38 chitinase 3-like 1 (CHI3L1) olfactomedin4 (OLFM4) and intelectin 1 were screened out between patients with IBD and controls When proteins with fold change gt12 orlt084 and P value lt 005 between groups were considered differentially expressed the expression of 10 proteins including CD38involved in the nicotinamide adenine dinucleotide (NAD) metabolism and signaling pathway showed significant changes in IBDUsing the NCBI GEO database we confirmed increased CD38 mRNA expression in patients with UC and in mouse colitis modelsProtein CD38 expression was higher in CD and UC than in normal controls CD38 expression was higher in inflamed tissues thanin noninflamed tissues and CD38 was located in F480-positive cells Our study may provide novel insights into the molecularpathogenesis of IBD Further studies are required on the role of NAD metabolism and CD38 in intestinal inflammation

1 Introduction

Inflammatory bowel disease (IBD) is categorized into Crohnrsquosdisease (CD) and ulcerative colitis (UC) which are char-acterized by relapsing chronic colitis in the gastrointestinaltract An estimated 25 million people are affected by IBD inEurope [1] In Asia although the prevalence of IBD is lowerthan that in Europe it has rapidly increased over the lastdecade [2 3]Thus IBD has become amajor health challengeworldwide However the precise etiological factors of IBDremain unclear Currently IBD is thought to result from

interplay between environmental factors and host geneticsleading to persistent gastrointestinal immune activation [45]

Various inflammatory molecules including cytokineschemokines and danger-associated molecular patterns(DAMPs) are released from infiltrating inflammatory cells[4] and drugs targeting these inflammatory molecules aredeveloped as therapeutics for IBD treatment [6] Tumornecrosis factor-120572 (TNF-120572) inhibitors are now the mostcommonly prescribed biologic therapeutics for patients withIBD Other new therapeutic concepts such as Janus kinase

HindawiBioMed Research InternationalVolume 2019 Article ID 3950628 11 pageshttpsdoiorg10115520193950628

2 BioMed Research International

(JAK) inhibitors antiadhesion molecules and anti-Smad7have shown promising results in current clinical trials [7ndash9]Much of the recent research in IBD has been focused onidentifying novel molecules that may be therapeutic targets

Currently IBDdiagnosis depends on clinical endoscopicradiographic and laboratory findings The differential diag-nosis of CD and UC is clear in most cases however itis difficult to determine in an estimated 15 of patientsbecause of atypical findings [10 11] Accurate diagnosis ofIBD and differential diagnosis between UC and CD areessential for ensuring appropriate therapeutic interventionand surveillance [12] Serological markers especially per-inuclear antineutrophil cytoplasmic antibodies (pANCAs)and antindashSaccharomyces cerevisiae antibodies (ASCAs) aidin differentiating UC from CD [13] however the sensitivityof this test is relatively low [14] Histological biomarkers forthis differential diagnosis are notwell understood Identifyingmolecules differentially expressed between CD and UC mayhelp uncover the differences in their pathogenesis

Proteomics helps provide novel strategies for large-scaleprotein identification analysis and valuable insights into dis-ease pathophysiology In the past decade proteomic inquirieshave helped uncover numerous host proteins and pathwaysrelated to IBD pathogenesis Utilizing matrix-assisted laserdesorptionionization (MALDI)ndashtime-of-flight (TOF) massspectrometry (MS) Anna et al [15] identified annexin A2and programmed cell death protein 8 as being involved inthe destruction of intestinal epithelial cell (IEC) homeostasisin UC Zhao et al [16] identified the p38 mitogen-activatedprotein kinase (MAPK) pathway as a molecular signaturein UC Moreover serum proteomic panels have been usedto differentiate CD from UC [17] to predict disease activity[18] and to evaluate response to infliximab (IFX) therapy[19] In the current study we aimed to identify potentialproteins involved in IBDpathophysiology and to compare theproteomic differences between CD and UC by using tandemmass tag- (TMT-) based quantitative proteomics in orderto identify novel proteins that may be associated with thepathogenesis of IBD and differentiation between CD andUC

2 Materials and Methods

21 Sample Collection The diagnostic criteria for both UCand CD were based on clinical endoscopic and histologicalfeatures according to the World Gastroenterology Organiza-tion Practice Guidelines for IBD diagnosis and management[20] For proteomic analysis patients with CD (n = 9) or UC(n = 9) were recruited from inpatients of the Departmentof Gastroenterology the First Affiliated Hospital of ZhejiangUniversity During colonoscopy two intestinal tissue biopsysamples were obtained from the inflamed area The normalcontrols were patients who underwent screening colono-scopies without active gastrointestinal pathology Age andsex-matched normal control patients (n = 6) were recruitedand samples were obtained from the normal colon tissueduring screening colonoscopy

The independent groups established for validation wereas follows surgically resected colon tissues from three controlpatients three patients with CD and three patients with UC

For patients with CD or UC both inflamed and noninflamedtissue samples were obtained Information regarding base-line clinical characteristics was obtained during admissionInformed consent was obtained from all subjects beforeparticipation The study protocol conforms to the ethicalguidelines of the 1975 Declaration of Helsinki (6th revision2008) as reflected in the approval by the ethics committee ofthe First Affiliated Hospital of Zhejiang University School ofMedicine In all cases colonoscopy biopsy or resected colontissue specimens were rinsed in phosphate-buffered salineand immediately frozen in liquid nitrogen before storage at-80∘C

22 Protein Extraction and Digestion Tissues were homoge-nized in radioimmunoprecipitation assay (RIPA) lysis bufferand were centrifuged Protein concentrations were measuredwith the bicinchoninic acid (BCA) assay (Beyotime BeijingChina) All sampleswere reducedwith dithiothreitol (10mM)at 60∘C for 30 min and alkylated with iodoacetamide (30mM) for 30 min at room temperature in the dark The pro-teinswere then incubatedwith cold acetone for 4 h at 0∘CTheprotein pellets were centrifuged at 3000 rpm for 15min at 4∘Cand resuspended in 50 120583M triethylammonium bicarbonate(TEAB) Trypsin (Thermo Fisher Scientific America) wasadded at a 150 enzyme substrate ratio for overnight digestionat 37∘C For TMT labeling 24 samples each containing 30120583g protein digest were divided into three TMT experimentsA common reference sample created by equal mixing of allsamples was labeled with TMT-131 and TMT-130C across all3 TMT experiments The TMT-labeling design is shown inSupplementary Table 1

23 Quantitative Proteomic Analysis TMT-labeled peptides(Thermo Fisher Scientific) were fractionated using the HPRPmethod and desalted before LC-MSMS analyses Technicaldetails regarding instrument parameters and operational pro-cesses can be found in Supplementary Materials and Meth-ods The RAW files acquired were loaded into MaxQuant(version 1610) and searched against the human UniProtKBdatabase (88473 sequences version 09-2015) Andromedawas used as search engine for the identification of proteinsThe database search was performed using the MS2 reporterion mode with the 10plex TMT option A mass toleranceof 7 ppm was set for the main database search Trypsinwith up to two missed cleavages was set Oxidation (M)and carbamidomethyl (C) were set as variable and fixedmodifications respectively An automatic decoy databasesearch was performed A protein level false-discovery rate(FDR) of 1 was set to filter the results

For quantitative analysis the TMT reporter ion intensityof each protein was first normalized against the medianintensity of all proteins within each sample to correct label-to-label variations Subsequently it was normalized againstthe averaged reference ion intensities of 131N and 131C labelswithin each run to correct run-to-run variations At leasttwo unique peptides were required for protein quantitationProteins with empty values were discarded Studentrsquos t-testwas performed for each protein between groups with thePerseus software Proteins that showed more than twofold

BioMed Research International 3

change (fold change of gt2 or lt05) with P value lt 005were considered to show significant differential expressionWhen analyzing proteins involved in nicotinamide adeninedinucleotide (NAD) metabolism and signaling pathwaysproteins that showed more than 12-fold change (fold changeof gt12 or lt084) were considered to show significant changeMultivariate principal component analysis (PCA) and heatmaps were used to summarize and visualize sample classifi-cation on the basis of expression profiles of all proteins

24 Bioinformatics Analysis ProteinAtlas was used to ana-lyze the tissue specificity of proteins differentially expressedbetween patients with IBD and the controls Differen-tially expressed proteins (DEPs) were then divided into 2groups gastrointestinal (GI) tissue-specific group (GI tis-sue types) and GI tissue-nonspecific group (other tissuetypes) Subsequently reactome pathway analysis (httpswwwreactomeorg) was used to cluster the pathways inwhich the 2 groups were involved The pathway analysisresults were visualized using a bubble chart CD38 geneexpression levels of patients with UC (datasets GDS3119 andGDS2642) and of mouse colitis models (datasets GDS4363and GDS3859) were downloaded from the NCBI GEOdatabaseThe expression data were analyzed with the Graph-Pad Prism 6 software

25 Mouse Model of Dextran Sulfate Sodium Salt-InducedColitis 12 male C57BL6J mice aged 6ndash8 weeks were pur-chased from the Zhejiang Academy of Medical Science Themice were orally administered 4 dextran sulfate sodium salt(DSS molecular weight 36000ndash50000 MP BiomedicalsSanta Ana CA USA) in water for 5 days to induce acutecolitis (n = 6group) Control mice (n = 6group) weregiven drinking water Body weight and stool consistencywere recorded every day On the sixth day the mice wereeuthanized with 5 chloral hydrateThe colons were resectedand fixed immediately in 10 formalin and embedded Thisstudy was performed according to the guidelines of theanimal ethics committee of the First Affiliated Hospital ofZhejiang University School of Medicine

26 Immunohistochemical Staining Immunohistochemical(IHC) staining was performed on paraffin-embedded sec-tions of patients who had undergone colon resection Tissueswere cut into 5 120583m-thick sections and stained with HampEbefore IHC staining Slides were incubated with primaryantibodies against ITLN1 (ab118232 Abcam) and CD38(ab108403 Abcam) for humans and CD38 (sc374650 SantaCruz) for mice overnight at 4∘C followed by incubation withhorseradish peroxidase-conjugated anti-IgG for 1 h at 37∘CThe samples were visualized with 331015840-diaminobenzidine(DAB) and observed under a light microscope (Leica Ger-many)

27 Immunofluorescence Staining Paraffin-embedded sec-tions of colon tissues from patients with IBD or controlswere usedThe slides were incubatedwith primary antibodiesagainst CD38 (sc374650 Santa Cruz) and F480 (ab16911

Abcam) at 4∘C overnight On the next day the slides wereincubated with sheep anti-mouse Alexa Fluor 488 (DaWenBiotech China) and goat anti-rat Cy3 (DaWen BiotechChina) antibodies at 37∘C for 1 h followed by incubationwith410158406-diamidino-2-phenylindole (DAPI) for 3 min Imageswere obtained using a laser scanning microscope (OlympusJapan)

28 Western Blot Analysis Specimens were homogenized inRIPA buffer and centrifuged at 12000 rpm for 15 min at4∘C subsequently the supernatants were collected Proteins(40 120583g) were separated using 10 sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis (PAGE) gels andwere then transblotted onto 045 120583m nitrocellulose mem-branes (Millipore Merck Germany) The membranes wereblocked with 5 nonfat milk Primary antibodies againstCD38 (ab108403 Abcam) and glyceraldehyde 3-phosphatedehydrogenase (GAPDH 5174 Cell Signaling Technology)were used After incubation with the secondary antibody thebands were visualized using an electrochemiluminescence(ECL) imaging system

29 Statistical Analysis Experimental data have beenexpressed in terms of mean plusmn standard error of the mean(SEM) values and the GraphPad Prism 6 software was usedfor comparison between groups Unpaired Studentrsquos t-testwas used to compare differences between two groups Formore than 2 groups analysis of variance (ANOVA) was usedA two-sided P value lt005 was considered to be statisticallysignificant

3 Results

31 Proteins Differentially Expressed between Controls andPatients with IBD We enrolled 6 controls 9 patients withCD and 9 patients with UC Using a protein level FDR of 1as a criterion 5702 5544 and 5552 proteins were identifiedfrom 3 TMT experiments (Figure 1(a)) A total of 4786proteins were identified from the 3 experiments of these 102were differentially expressed between patients with IBD andcontrols The proteome between patients with IBD and con-trols could be clearly separated as visualized by the PCA plot(Figure 1(b)) and heat map (Figure 1(c)) Among the DEPs59 proteins showed higher expression and 43 showed lowerexpression in patients with IBD than in controls (Figure 2(a))Seventeen proteins were differentially expressed betweenpatients with CD and patients with UC (Figure 2(b)) To ana-lyze the related functions of proteins differentially expressedbetween patients with IBD and controls reactome clusteringpathway analysis was performed Pathways enriched by DEPsfrom other tissues were mainly involved in the immunesystem including the adaptive immune system and processessuch as antigen presentation interferon alphabeta signalingand the neutrophil degranulation pathway (Figure 2(c))Pathways enriched by DEPs from GI tissue types includedthe mRNA splicing-major pathway pyruvate metabolismand citric acid (TCA) cycle gene and protein expression


TMT15702

TMT25544

TMT35552

4786

329

192 187

287 300

279

(a)

80

60

40

20

0

minus20

minus40

minus60

minus80

t[2]

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

t[1]NCUCCD

(b)

-3

+3

0

CDUCNC

N_2

N_6

N_3

N_4

N_1

N_5

UC_8

UC_15

UC_12

CD_19

CD_21

CD_20

CD_24

CD_17

CD_18

CD_16

CD_23

CD_22

UC_9

UC_14

UC_13

UC_7

UC_10

UC_11

(c)

Figure 1 Summary of proteomics analysis ofNCCD andUCusingTMTquantitationmethod (a) Venn diagram showing proteins identifiedacross 3 TMT experiments from which 4786 commonly identified proteins were used for downstream analyses (b) Overall differences ofproteome between NC CD and UC were summarized by PCA plot (c) Heat map representation of abundance profiles of all 4786 proteinsin all samples Color shade correlates with relative protein abundances across each row (redgreen for upregulationdownregulation)


0

1

2

3

4

5

6

7

8

minus3 minus2 minus1 0 1 2 3

IBD vs NC

P lt 005

43 Significantlydown-regulated

proteins


proteins

Log2 Fold Change

-Log

10P-

valu

e

ITLN1

OLFM4

CD38

CHI3L1

(a)

0

05

1

15

2

25

3

35

4


CD vs UC

P lt 005


proteins


proteins

Log2 Fold Change

-Log

10P-

valu

e

ACE ACE2

(b)

-Log

10 P

val

ue

Pathways enriched by DEP from other tissue types

Ratio of enrichedtotal genes in the pathway

18

16

14

12

10

8

6

4

2

0

0 001 002 003 004 005

(c)

-Log

10 P

val

ue

Pathways enriched by DEP from GI tissue types

Ratio of enrichedtotal genes in the pathway0 001 002 003 004 005

8

7

6

5

4

3

2

1

(d)

Figure 2 Identification and pathway analysis of DEPs (a) Volcano map of DEPs between IBD and NC (b) Volcano map of DEPs betweenCD and UC (c) Reactome pathway analysis of DEPs enriched from no tissue types (d) Reactome pathway analysis of DEPs enriched fromGI tissue types

by JAK-STAT signaling after interleukin-12 stimulation andapoptosis pathway (Figure 2(d))

32 DEPs according to Disease Subtypes of InflammatoryBowel Disease 102 proteins were significantly dysregulatedin patients with IBD (Figure 2(a)) Of these several pro-teins have been used as disease markers Among themlipocalin 2 (LCN2) S100A12 and matrix metallopeptidase 9(MMP9)were upregulated in bothUCandCDwhile S100A8S100A9 myeloperoxidase (MPO) and lactotransferrin (LTF)were upregulated in CD (Supplementary Table 2) Severalnovel proteins were also identified (Table 1) Chitinase 3-like 1 (CHI3L1) CD38 molecule (CD38) and olfactomedin4 (OLFM4) were upregulated in patients with CD or UCwhereas intelectin 1 (ITLN1) was downregulated We alsoidentified proteins that were differentially expressed betweenthe two subtypes of IBDOur results indicated that 17 proteinswere upregulated in CD compared to UC (Figure 2(b)) Someof the proteins with abundance changes are shown in Table 2from these angiotensin converting enzyme 2 (ACE2) and

angiotensin converting enzyme 1 (ACE) showed significantlyhigher expression in CD than in UC

33 NAD Metabolism and Signaling Pathway Showed Alter-ations in Patients with IBD Using reactome pathway anal-ysis we noted that many proteins are involved in immunepathways Previous studies have revealed that CD38 playsmultiple functions in rheumatoid arthritis (RA) allergicairway disease andmultiple myeloma and is expressed in themembrane of immune cells [21ndash23] Furthermore Michaelreported that CD38 is expressed on inflammatory cells of theintestine and promotes intestinal inflammation [24] There-fore we studied the role of CD38 in IBD CD38 participatesin the synthesis of cyclic ADP ribose (cADPR) from NADand the NAD metabolism pathway is reported to promoteinflammation in the gut [24 25] therefore we analyzedour proteomic results for the expression of other moleculesinvolved in NAD+ metabolism and signaling Twenty-twoproteins involved in NAD metabolism and signaling wereidentified by the proteomic analysis (Table 3) When the


AOX1

CD38

QPRT

NT5E

NMRK12NMNAT1

PNP

NAPRT

NMRK12

NAMPT

CD38

NADK

NNT

NADSYN1

Nicotinuate NA (VB3) NAR

NAM

NR

N1-Methyl-nicotinamide

N1-Methyl-4-pyridone-5-carboxamide


NNMT

NADP

NAD

NaADNaMN

QA

Amino acid Metabolism

NADK2

PNP

NT5C1BNT5C3B

NMNAT2NMNAT3

NMNAT1NMNAT2NMNAT3

NT5C2NT5C NT5C3A

NT5MNT5C1A

NUDT12

ENPP13

NUDT12

ASPDH

Upregulated in IBD

Downregulated in IBD

Unchanged

ENPP13

Expression Trends

NAD metabolism and signaling

NAD+ Salvage pathway

NAD De novo synthesis

NAD Degradation

cADPRCD38

NAADPCD38

NAADPR

RYR

Inflammationapoptosis

NMN

Significantly (plt005) upregulated

Significantly (plt005) downregulated

Metabolites

SIRT1NFKB1NFKB2

ADPR

PARP9PARP14

ERSR Ca2+ signaling

Undetected

Figure 3The role and expression of proteins identified by proteomic analysis within NAD+metabolism and signaling pathway Red (green)rectangle represents proteins upregulated (downregulated) in IBD Red (green) rectangle with red pentagram represents proteins upregulated(downregulated) in IBDwith p valuelt 005 Yellow rectangle represents proteins that are unchanged between IBDand controls Gray rectanglerepresents proteins undetected by proteomic analysis

Table 1 Novel proteins showing abundance changes in CD or UC patients

UniProt accession Protein UCCon CDCon CDUC Unique peptides GI related or notFold change p value Fold change p value Fold change p value

P36222 CHI3L1 342 00434 313 00048 092 07712 4 notP28907 CD38 228 00015 270 00004 118 02881 9 notQ6UX06 OLFM4 232 00779 189 00030 081 04557 5 notQ8WWA0 ITLN1 050 00048 036 00000 073 01718 6 yesCD Crohnrsquos disease UC ulcerative colitis Con control GI gastrointestinal

fold change level was set at gt12 or lt084 10 of themshowed significant change (Table 3) most of these wereenzymes related to NAD synthesis and cleavageThe role andexpression of these proteins within the NADmetabolism andsignaling pathway are indicated in Figure 3

34 CD38 Expression Increased in Patients with IBD and in theMouse Colitis Models We analyzed CD38 mRNA expressionby using the NCBI GEO database The expression data in theGDS3119 database indicated that the inflamed tissues in UChad higher CD38 expression than the controls (Figure 4(a))TheCD38 expression in the inflamed regions was higher thanthat in the noninflamed regions (Figures 4(a) and 4(b)) In theDSS-induced colitis model and T cell transport colitis modelCD38 expression gradually increased with the emergence ofcolitis (Figures 4(c) and 4(d))

We then analyzedCD38 protein expression in colon spec-imens frompatientswithCDorUCCD38protein expressionwas higher in patients with CD or UC than in controls(Figures 5(a) and 5(b)) The CD38 expression in inflamedregions was higher than that in noninflamed regions (Figures5(c) and 5(d)) IHC and immunofluorescence (IF) stainingconfirmed increased CD38 expression and membrane CD38distribution (Figures 5(e) and 5(f)) We noted colocalizationof CD38 with the macrophage marker F480 (Figure 5(f))CD38 protein expression also increased in themicewithDSS-induced colitis (Supplementary Figure 1)

4 Discussion

Since the first study by Barcelo-Batllori et al [26] whoidentified increased indoleamine-23-dioxygenase expression


0

2

4

6

8

10

GDS3119

NCUCNI

UCI

CD38

relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

lowastlowast

(a)

0

2

4

6

8

GDS2642

NCUCNI

UCI

CD38

relat

ive e

xpre

ssio

n

lowast

lowastns

(b)

GDS3859

0D 2D 4D 6D0

5000

10000

15000

CD38

relat

ive e

xpre

ssio

n

lowast

ns

lowastlowastlowast

(c)

GDS4363

NC 4W 6W

0

5000

10000

15000

20000

CD38

relat

ive e

xpre

ssio

n

lowast

lowast

(d)

Figure 4 CD38 mRNA expression in patients with UC and animal colitis models from GEO datasets (a) Colon mucosa CD38 mRNAexpression in NC UCI and UCNI patients based on GEO GDS3319 dataset (b) Colon mucosa CD38 mRNA expression in UCI and UCNIpatients based on GEO GDS2642 dataset (c) Colon CD38 mRNA expression in DSS induced mouse colitis model at 0D 2D 4D and 6Dbased on GEOGDS3859 dataset (d) Colon CD38mRNA expression in T cell transfer mouse colitis model at 0W 4W and 8W based on GEOGDS4363 dataset Significance level lowastP lt 005 lowastlowastP lt 001 and lowast lowast lowastP lt 0001 NC normal control CD Crohnrsquos disease UC ulcerativecolitis UCI ulcerative colitis inflamed UCNI ulcerative colitis noninflamed D day W week

Table 2 Proteins showing abundance changes between CD and UC patients


P05062 ALDOB 155 01156 862 00843 556 00477 17 notQ9BYF1 ACE2 130 02869 641 00575 491 00274 2 notP12104 FABP2 102 09457 487 00912 478 00394 9 notP12821 ACE 126 00372 396 00580 313 00324 12 notCD Crohnrsquos disease UC ulcerative colitis Con control GI gastrointestinal

in cytokine-treated colon epithelial cells by using proteomicstechnology numerous studies have investigated proteomicchanges in IBD We previously identified several proteinpeaks in relation to serum samples which were helpful fordifferentiating CD from intestinal tuberculosis (ITB) [27]Isobaric chemical labeling for quantitative proteomics hasbetter quantification performance and reproducibility thanother proteomic methods [28] In this study we employed

TMT-based quantitative proteomics to identify DEPs inpatients with IBD

We identified several previously reported proteins suchas S100A89 S100A12 LTF LCN2 andMMP9 most of whichare used to evaluate the disease activity of IBD [29ndash32] Wealso identified several novel proteins associated with IBDCH3L1 CD38 and OLFM4 showed increased levels whereasITLN1 showed decreased levels Previous DNA microarray


Table 3 Expression of proteins involved in NAD metabolism and signaling pathway within the proteomic results

UniProtaccession Protein p value Fold change Unique GI related or not

IBDCon peptides notP36222 CD38 00030 249 9 notO95544 NADK 00768 143 5 notQ4G0N4 NADK2 00183 082 6 notQ6IA69 NADSYN1 07181 097 3 notC9JF35 NAMPT 00084 177 14 notQ6XQN6 NAPRT 03029 088 14 notQ9HAN9 NMNAT1 0112 087 3 notQ96T66 NMNAT3 01096 079 2 notP40261 NNMT 00013 173 6 notQ13423 NNT 00023 079 4 yesQ8TCD5 NT5C 01245 083 6 notP49902 NT5C2 04623 094 4 notQ9H0P0 NT5C3A 00288 067 7 notP21589 NT5E 07597 092 3 notQ9BQG2 NUDT12 00875 082 2 notP00491 PNP 00131 134 10 notQ15274 QPRT 01998 147 4 notQ96EB6 SIRT1 01482 119 2 notQ8IXQ6 PARP9 00038 153 9 notQ460N5 PARP14 00013 152 12 yesQ00653 NFKB2 00446 165 4 notP19838 NFKB1 07749 102 11 notIBD inflammatory bowel disease Con control GI gastrointestinal

analysis has shown that CH3L1 is upregulated in inflamedmucosa [33] our result is consistent with these microarrayresults Several studies also revealed that fecal CHI3L1 aids inpredicting the severity and activity of intestinal inflammationin both pediatric and adult IBD [34 35] However fecalCHI3L1 analysis has not been analyzed in Asian populationsOLFM4 protein expression was found to increase by 17 foldsin CD and 37 folds in UC [36] which is similar to our resultsITLN1 is a lactoferrin receptor that can recognize microbialglycans in the intestine [37] Previous studies revealed thatserum ITLN1 levels decrease in IBD and are negativelycorrelated with its disease activity [38] However the role ofITLN1 in IBD pathogenesis is still unclear

Differential diagnosis between CD and UC is importantfor guiding treatment and follow-up In the current study weidentified 17 proteins that showed differences in expressionbetween CD and UC Among these ACE2 and ACE showedmuch higher expression in patients with CD than in patientswith UC Both ACE2 and ACE are associated with the devel-opment of organ fibrosis [39 40] and CD is characterized bysubepithelial fibrosis in some patients which might explainthe increased ACE2 and ACE levels in CD However thefunctions of the proteins identified were unclear and need tobe confirmed in future studies

Proteomic analysis indicated that the expression of manyproteins involved in NAD metabolism and signaling showed

changes suggesting that NAD metabolism and signaling areassociated with the gut inflammation noted in IBD NAD is amajor coenzyme in bioenergetic processes including oxida-tive phosphorylation and energy homeostasis [41] NAD isalso the substrate for NAD-cleaving enzymes such as poly(ADP-ribose) polymerases (PARPs) sirtuins (SIRTs) andcADP-ribose synthases such as CD38 [42ndash44] NAD cleav-age by these enzymes is important for many physiologicalprocesses NAD synthesis consists of two pathways the denovo synthesis pathway and salvage synthesis pathway withthe latter playing an important role in mammals In thesalvage pathway NAMPT is the key enzyme catalyzing NADsynthesis A previous proteomic study revealed that NAMPTlevels increase in the inflamed colonic mucosa of patientswith IBD [45] which was also confirmed by our studyRomana et al reported that the NAMPT inhibitor FK866alleviates the PARPSIRT-mediated inflammatory responseand alters macrophage polarization in DSS-induced colitisin mice [25] NAMPT inhibition leads to decreased CD38+immune cell infiltration into the inflamed colon Howeverthe roles of the other enzymes identified by our study in thepathogenesis of colitis are not clear

The CD38 expression level has been further confirmed byvalidation studies CD38 is an ectoenzyme that catalyzes thesynthesis of cADPR and NAADP from NAD+ [46] CD38-cADPR signaling can mediate airway hyperresponsiveness


Control UC

CD38

GAPDH

(a)

CD38

GAPDH

Control CD

(b)

3 I 3 NI1 I 1 NI 2 I 2 NI

CD38

GAPDH

(c)

3 NI1 I 1 NI 2 I 2 NI 3 I

CD38

GAPDH

(d)

Control

UC

CD

(e)

DAPI CD38 F480 Merged

Control

UC

CD

(f)

Figure 5 CD38 is increased in IBD patients (a) Western blot analysis of CD38 expression in control and UC patients (N=3 per group) (b)Western blot analysis of CD38 expression in 3 matched pairs of inflamed (I) and noninflamed (NI) UC tissues (c) Western blot analysis ofCD38 expression in control and CD patients (N=3 per group) (d)Western blot analysis of CD38 expression in 3matched pairs of inflamed (I)and noninflamed (NI) CD tissues (e) Expression of CD38 by IHC (original magnificationtimes100) (f) Immunofluorescence staining of DAPI(blue) CD38 (green) and F480 (red) in control UC and CD patients (N=3 per group) (original magnification times200) CD Crohnrsquos diseaseUC ulcerative colitis

by increasing calcium release in airway smooth muscle cells[22] CD38 is also involved in multiple myeloma antibodiesagainst CD38 including daratumumab and MOR202 arepromising therapeutics for multiple myeloma [23] Usingmicroarray analysis Chang et al [21] found that CD38increased in RA synovial tissues Recently a study usingRNA sequencing also revealed that CD38 was significantlyupregulated in the synovial tissue of patients with RA atvarious stages [47] Furthermore their ex vivo experimentsshowed that daratumumab effectively depletes plasma cellsin peripheral blood mononuclear cells (PBMCs) and thatCD38 inhibition can be a novel treatment option for bothRA and systemic lupus erythematosus (SLE) CD38minusminus micehave shown decreased immune cell infiltration and mildcolitis symptoms uponDSS treatment [24] Shu et al reportedthat CD38 expression increased in macrophages upon LPSstimulation and CD38 suppression inhibited macrophageM1polarization and activation of nuclear factor-120581B (NF-120581B) sig-naling [48] suggesting that CD38 expression inmacrophagesis proinflammatory Our results showed that CD38 was

localized in F480+ macrophages however we could notexclude the distribution of CD38 within other cell typesThe molecular mechanisms underlying the effect of CD38 inintestinal macrophages in colitis require further research

5 Conclusion

Using TMT proteomic quantification the current studyidentified proteins that were differentially expressed betweenpatients with IBD and controls We found that proteinsinvolved in the NAD metabolism and signaling pathwayshowed significant alterations in IBD of these the expressionof CD38 was validated Further studies are required to clarifythe mechanisms underlying the promotion of intestinalinflammation by CD38 and to determine whether CD38inhibition can be used as a treatment option for IBD

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request


Conflicts of Interest

The authors declare that there are no conflicts of interest

Acknowledgments

The authors thank Ming Yang and Lihua Chen for biopsysample collection and Huatuo Zhu for the technical assis-tance This work was supported by the Medical ScienceResearch Foundation of Health Bureau of Zhejiang Province(WKJ-ZJ-1516) the National Key Research and DevelopmentProgram (2017YFC1200100) and the National Natural Sci-ence Foundation of China (81400589)

Supplementary Materials

Supplementary 1 Materials and Methods technical detailsregarding the instrument parameters and operational processof TMT labeling high pH reversed phase fractionation andLC-MSMS analysisSupplementary 2 Table 1 the TMT-labeling design forcontrols patients with UC and patients with CDSupplementary 3 Table 2 proteins used as disease markersand upregulated in UC or CDSupplementary 4 Figure 1 CD38 protein expressionincreased in mice with DSS-induced colitisSupplementary 5 Excel files quantitation values significancein the different comparisons performed peptides identifiedand so forth

References

[1] J Burisch T Jess M Martinato and P L Lakatos ldquoThe burdenof inflammatory bowel disease in Europerdquo Journal of Crohnrsquosand Colitis vol 7 no 4 pp 322ndash337 2013

[2] L Prideaux M A Kamm P P De Cruz F K L Chan and S CNg ldquoInflammatory bowel disease in Asia a systematic reviewrdquoJournal of Gastroenterology and Hepatology vol 27 no 8 pp1266ndash1280 2012

[3] S C Ng H Y Shi N Hamidi et al ldquoWorldwide incidence andprevalence of inflammatory bowel disease in the 21st century asystematic review of population-based studiesrdquoThe Lancet vol390 no 10114 pp 2769ndash2778 2017

[4] J H Park L Peyrin-Biroulet M Eisenhut and J I Shin ldquoIBDimmunopathogenesis A comprehensive review of inflamma-tory moleculesrdquo Autoimmunity Reviews vol 16 no 4 pp 416ndash426 2017

[5] R J Xavier and D K Podolsky ldquoUnravelling the pathogenesisof inflammatory bowel diseaserdquo Nature vol 448 no 7152 pp427ndash434 2007

[6] M F Neurath ldquoNew targets for mucosal healing and therapy ininflammatory bowel diseasesrdquoMucosal Immunology vol 7 no1 pp 6ndash19 2014

[7] W J Sandborn S Ghosh J Panes et al ldquoTofacitinib an oralJanus kinase inhibitor in active ulcerative colitisrdquo The NewEngland Journal of Medicine vol 367 no 7 pp 616ndash624 2012

[8] W J Sandborn B G Feagan and P Rutgeerts ldquoVedolizumabas induction and maintenance therapy for Crohnrsquos diseaserdquoThe

New England Journal of Medicine vol 369 no 8 pp 711ndash7212013

[9] G Monteleone M F Neurath S Ardizzone et al ldquoMongersenan oral SMAD7 antisense oligonucleotide and crohnrsquos diseaserdquoThe New England Journal of Medicine vol 372 no 12 pp 1104ndash1113 2015

[10] K Geboes J-F Colombel A Greenstein et al ldquoIndeterminatecolitis A review of the concept - Whatrsquos in a namerdquo Inflamma-tory Bowel Diseases vol 14 no 6 pp 850ndash857 2008

[11] W J Tremaine ldquoIs indeterminate colitis determinablerdquoCurrentFungal Infection Reports vol 14 no 2 pp 162ndash165 2012

[12] M Farmer R E Petras L E Hunt J E Janosky and SGalandiuk ldquoThe importance of diagnostic accuracy in colonicinflammatory bowel diseaserdquo American Journal of Gastroen-terology vol 95 no 11 pp 3184ndash3188 2000

[13] M Ferrante L Henckaerts M Joossens et al ldquoNew serologicalmarkers in inflammatory bowel disease are associated withcomplicated disease behaviourrdquo Gut vol 56 no 10 pp 1394ndash1403 2007

[14] G E Reese V A Constantinides C Simillis et al ldquoDiagnosticprecision of anti-Saccharomyces cerevisiae antibodies and per-inuclear antineutrophil cytoplasmic antibodies in inflammatorybowel diseaserdquo American Journal of Gastroenterology vol 101no 10 pp 2410ndash2422 2006

[15] A Shkoda TWerner H Daniel M Gunckel G Rogler and DHaller ldquoDifferential protein expression profile in the intestinalepithelium from patients with inflammatory bowel diseaserdquoJournal of Proteome Research vol 6 no 3 pp 1114ndash1125 2007

[16] X Zhao B Kang C Lu et al ldquoEvaluation of P38 MAPKpathway as a molecular signature in ulcerative colitisrdquo Journalof Proteome Research vol 10 no 5 pp 2216ndash2225 2011

[17] M Hatsugai M S Kurokawa T Kouro et al ldquoProtein profilesof peripheral bloodmononuclear cells are useful for differentialdiagnosis of ulcerative colitis and Crohnrsquos diseaserdquo Journal ofGastroenterology vol 45 no 5 pp 488ndash500 2010

[18] R Burakoff V Pabby L Onyewadume et al ldquoBlood-basedbiomarkers used to predict disease activity in crohnrsquos diseaseand ulcerative colitisrdquo Inflammatory Bowel Diseases vol 21 no5 pp 1132ndash1140 2015

[19] M Gazouli A K Anagnostopoulos A Papadopoulou et alldquoSerum protein profile of Crohnrsquos disease treated with inflix-imabrdquo Journal of Crohnrsquos and Colitis vol 7 no 10 pp e461ndashe4702013

[20] C N Bernstein M Fried J H Krabshuis et al ldquoWorld gas-troenterology organization practice guidelines for the diagnosisandmanagement of IBD in 2010rdquo Inflammatory Bowel Diseasesvol 16 no 1 pp 112ndash124 2010

[21] X Chang L Yue W Liu et al ldquoCD38 and E2F transcriptionfactor 2 have uniquely increased expression in rheumatoidarthritis synovial tissuesrdquoClinical amp Experimental Immunologyvol 176 no 2 pp 222ndash231 2014

[22] D A Deshpande A G P Guedes F E Lund S SubramanianT F Walseth and M S Kannan ldquoCD38 in the pathogenesis ofallergic airway disease Potential therapeutic targetsrdquo Pharma-cology ampTherapeutics vol 172 pp 116ndash126 2017

[23] N W C J Van De Donk P G Richardson and F MalavasildquoCD38 antibodies in multiple myeloma Back to the futurerdquoBlood vol 131 no 1 pp 13ndash29 2018

[24] M Schneider V Schumacher T Lischke et al ldquoCD38 isexpressed on inflammatory cells of the intestine and promotesintestinal inflammationrdquo Plos One vol 10 no 5 Article IDe0126007 2015


[25] R R Gerner V Klepsch S Macheiner et al ldquoNADmetabolismfuels human andmouse intestinal inflammationrdquoGut pp 1813ndash1823 2017

[26] S Barcelo-Batllori M Andre C Servis et al ldquoProteomicanalysis of cytokine induced proteins in human intestinalepithelial cells Implications for inflammatory bowel diseasesrdquoProteomics vol 2 no 5 pp 551ndash560 2002

[27] F Zhang C Xu L Ning et al ldquoExploration of serum proteomicprofiling and diagnostic model that differentiate crohnrsquos diseaseand intestinal tuberculosisrdquo Plos One vol 11 no 12 Article IDe0167109 2016

[28] Z Li R M Adams K Chourey G B Hurst R L Hettich andC Pan ldquoSystematic comparison of label-free metabolic label-ing and isobaric chemical labeling for quantitative proteomicson LTQ orbitrap velosrdquo Journal of Proteome Research vol 11 no3 pp 1582ndash1590 2012

[29] S Ikhtaire M S Shajib W Reinisch and W I Khan ldquoFecalcalprotectin its scope and utility in the management of inflam-matory bowel diseaserdquo Journal of Gastroenterology vol 51 no5 pp 434ndash446 2016

[30] J Stallhofer M Friedrich A Konrad-Zerna et al ldquoLipocalin-2 is a disease activity marker in inflammatory bowel diseaseregulated by IL-17A IL-22 and TNF-120572 andmodulated by IL23Rgenotype statusrdquo Inflammatory Bowel Diseases vol 21 no 10pp 2327ndash2340 2015

[31] K-L Kolho T Sipponen E Valtonen and E Savilahti ldquoFecalcalprotectin MMP-9 and human beta-defensin-2 levels inpediatric inflammatory bowel diseaserdquo International Journal ofColorectal Disease vol 29 no 1 pp 43ndash50 2014

[32] A A Soubieres and A Poullis ldquoEmerging biomarkers for thediagnosis and monitoring of inflammatory bowel diseasesrdquoInflammatory Bowel Diseases vol 22 no 8 pp 2016ndash2022 2016

[33] C-C Chen J Pekow V Llado et al ldquoChitinase 3-like-1 expres-sion in colonic epithelial cells as a potentially novel marker forcolitis-associated neoplasiardquoTheAmerican Journal of Pathologyvol 179 no 3 pp 1494ndash1503 2011

[34] T Aomatsu H Imaeda K Matsumoto et al ldquoFaecal chitinase3-like-1 A novel biomarker of disease activity in paediatricinflammatory bowel diseaserdquoAlimentary PharmacologyampTher-apeutics vol 34 no 8 pp 941ndash948 2011

[35] A Buisson E Vazeille R Minet-Quinard et al ldquoFaecalchitinase 3-like 1 is a reliable marker as accurate as faecalcalprotectin in detecting endoscopic activity in adult patientswith inflammatory bowel diseasesrdquo Alimentary PharmacologyampTherapeutics vol 43 no 10 pp 1069ndash1079 2016

[36] M Gersemann S Becker S Nuding et al ldquoOlfactomedin-4 isa glycoprotein secreted into mucus in active IBDrdquo Journal ofCrohnrsquos and Colitis vol 6 no 4 pp 425ndash434 2012

[37] D A Wesener K Wangkanont R McBride et al ldquoRecognitionof microbial glycans by human intelectin-1rdquo Nature Structuralamp Molecular Biology vol 22 no 8 pp 603ndash610 2015

[38] Y Lu L Zhou L Liu et al ldquoSerum omentin-1 as a diseaseactivity marker for crohnrsquos diseaserdquo Disease Markers vol 2014Article ID 162517 2014

[39] A HiroseMOno T Saibara et al ldquoAngiotensin II type 1 recep-tor blocker inhibits fibrosis in rat nonalcoholic steatohepatitisrdquoHepatology vol 45 no 6 pp 1375ndash1381 2007

[40] C H Osterreicher K Taura S De Minicis et al ldquoAngiotensin-converting-enzyme 2 inhibits liver fibrosis inmicerdquoHepatologyvol 50 no 3 pp 929ndash938 2009

[41] C Canto K J Menzies and J Auwerx ldquoNAD+ metabolismand the control of energy homeostasis a balancing act betweenmitochondria and the nucleusrdquo Cell Metabolism vol 22 no 1pp 31ndash53 2015

[42] V Schreiber F Dantzer J C Ame and G de MurcialdquoPoly(ADP-ribose) novel functions for an old moleculerdquoNature Reviews Molecular Cell Biology vol 7 no 7 pp 517ndash5282006

[43] N M Borradaile and G Pickering ldquoNAD+ sirtuins andcardiovascular diseaserdquo Current Pharmaceutical Design vol 15no 1 pp 110ndash117 2009

[44] S Partida-Sanchez A Gasser R Fliegert et al ldquoChemotaxisof mouse bone marrow neutrophils and dendritic cells iscontrolled by ADP-ribose the major product generated by theCD38 enzyme reactionrdquoThe Journal of Immunology vol 179 no11 pp 7827ndash7839 2007

[45] A E Starr S A Deeke Z Ning et al ldquoProteomic analysisof ascending colon biopsies from a paediatric inflammatorybowel disease inception cohort identifies protein biomarkersthat differentiate Crohnrsquos disease from UCrdquo Gut vol 66 no 9pp 1573ndash1583 2017

[46] F E Lund D A Cockayne T D Randall N Solvason FSchuber and M C Howard ldquoCD38 A new paradigm inlymphocyte activation and signal transductionrdquo ImmunologicalReviews vol 161 pp 79ndash93 1998

[47] S Cole A Walsh X Yin et al ldquoIntegrative analysis revealsCD38 as a therapeutic target for plasma cell-rich pre-diseaseand established rheumatoid arthritis and systemic lupus erythe-matosusrdquoArthritis ResearchampTherapy vol 20 no 1 p 85 2018

[48] B Shu Y Feng Y Gui et al ldquoBlockade of CD38 diminisheslipopolysaccharide-induced macrophage classical activationand acute kidney injury involving NF-120581B signaling suppres-sionrdquo Cellular Signalling vol 42 pp 249ndash258 2018

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


(JAK) inhibitors antiadhesion molecules and anti-Smad7have shown promising results in current clinical trials [7ndash9]Much of the recent research in IBD has been focused onidentifying novel molecules that may be therapeutic targets

Currently IBDdiagnosis depends on clinical endoscopicradiographic and laboratory findings The differential diag-nosis of CD and UC is clear in most cases however itis difficult to determine in an estimated 15 of patientsbecause of atypical findings [10 11] Accurate diagnosis ofIBD and differential diagnosis between UC and CD areessential for ensuring appropriate therapeutic interventionand surveillance [12] Serological markers especially per-inuclear antineutrophil cytoplasmic antibodies (pANCAs)and antindashSaccharomyces cerevisiae antibodies (ASCAs) aidin differentiating UC from CD [13] however the sensitivityof this test is relatively low [14] Histological biomarkers forthis differential diagnosis are notwell understood Identifyingmolecules differentially expressed between CD and UC mayhelp uncover the differences in their pathogenesis

Proteomics helps provide novel strategies for large-scaleprotein identification analysis and valuable insights into dis-ease pathophysiology In the past decade proteomic inquirieshave helped uncover numerous host proteins and pathwaysrelated to IBD pathogenesis Utilizing matrix-assisted laserdesorptionionization (MALDI)ndashtime-of-flight (TOF) massspectrometry (MS) Anna et al [15] identified annexin A2and programmed cell death protein 8 as being involved inthe destruction of intestinal epithelial cell (IEC) homeostasisin UC Zhao et al [16] identified the p38 mitogen-activatedprotein kinase (MAPK) pathway as a molecular signaturein UC Moreover serum proteomic panels have been usedto differentiate CD from UC [17] to predict disease activity[18] and to evaluate response to infliximab (IFX) therapy[19] In the current study we aimed to identify potentialproteins involved in IBDpathophysiology and to compare theproteomic differences between CD and UC by using tandemmass tag- (TMT-) based quantitative proteomics in orderto identify novel proteins that may be associated with thepathogenesis of IBD and differentiation between CD andUC

2 Materials and Methods

21 Sample Collection The diagnostic criteria for both UCand CD were based on clinical endoscopic and histologicalfeatures according to the World Gastroenterology Organiza-tion Practice Guidelines for IBD diagnosis and management[20] For proteomic analysis patients with CD (n = 9) or UC(n = 9) were recruited from inpatients of the Departmentof Gastroenterology the First Affiliated Hospital of ZhejiangUniversity During colonoscopy two intestinal tissue biopsysamples were obtained from the inflamed area The normalcontrols were patients who underwent screening colono-scopies without active gastrointestinal pathology Age andsex-matched normal control patients (n = 6) were recruitedand samples were obtained from the normal colon tissueduring screening colonoscopy

The independent groups established for validation wereas follows surgically resected colon tissues from three controlpatients three patients with CD and three patients with UC

For patients with CD or UC both inflamed and noninflamedtissue samples were obtained Information regarding base-line clinical characteristics was obtained during admissionInformed consent was obtained from all subjects beforeparticipation The study protocol conforms to the ethicalguidelines of the 1975 Declaration of Helsinki (6th revision2008) as reflected in the approval by the ethics committee ofthe First Affiliated Hospital of Zhejiang University School ofMedicine In all cases colonoscopy biopsy or resected colontissue specimens were rinsed in phosphate-buffered salineand immediately frozen in liquid nitrogen before storage at-80∘C

22 Protein Extraction and Digestion Tissues were homoge-nized in radioimmunoprecipitation assay (RIPA) lysis bufferand were centrifuged Protein concentrations were measuredwith the bicinchoninic acid (BCA) assay (Beyotime BeijingChina) All sampleswere reducedwith dithiothreitol (10mM)at 60∘C for 30 min and alkylated with iodoacetamide (30mM) for 30 min at room temperature in the dark The pro-teinswere then incubatedwith cold acetone for 4 h at 0∘CTheprotein pellets were centrifuged at 3000 rpm for 15min at 4∘Cand resuspended in 50 120583M triethylammonium bicarbonate(TEAB) Trypsin (Thermo Fisher Scientific America) wasadded at a 150 enzyme substrate ratio for overnight digestionat 37∘C For TMT labeling 24 samples each containing 30120583g protein digest were divided into three TMT experimentsA common reference sample created by equal mixing of allsamples was labeled with TMT-131 and TMT-130C across all3 TMT experiments The TMT-labeling design is shown inSupplementary Table 1

23 Quantitative Proteomic Analysis TMT-labeled peptides(Thermo Fisher Scientific) were fractionated using the HPRPmethod and desalted before LC-MSMS analyses Technicaldetails regarding instrument parameters and operational pro-cesses can be found in Supplementary Materials and Meth-ods The RAW files acquired were loaded into MaxQuant(version 1610) and searched against the human UniProtKBdatabase (88473 sequences version 09-2015) Andromedawas used as search engine for the identification of proteinsThe database search was performed using the MS2 reporterion mode with the 10plex TMT option A mass toleranceof 7 ppm was set for the main database search Trypsinwith up to two missed cleavages was set Oxidation (M)and carbamidomethyl (C) were set as variable and fixedmodifications respectively An automatic decoy databasesearch was performed A protein level false-discovery rate(FDR) of 1 was set to filter the results

For quantitative analysis the TMT reporter ion intensityof each protein was first normalized against the medianintensity of all proteins within each sample to correct label-to-label variations Subsequently it was normalized againstthe averaged reference ion intensities of 131N and 131C labelswithin each run to correct run-to-run variations At leasttwo unique peptides were required for protein quantitationProteins with empty values were discarded Studentrsquos t-testwas performed for each protein between groups with thePerseus software Proteins that showed more than twofold










3 Results



TMT15702

TMT25544

TMT35552

4786

329

192 187

287 300

279

(a)

80

60

40

20

0

minus20

minus40

minus60

minus80

t[2]


t[1]NCUCCD

(b)

-3

+3

0

CDUCNC

N_2

N_6

N_3

N_4

N_1

N_5

UC_8

UC_15

UC_12

CD_19

CD_21

CD_20

CD_24

CD_17

CD_18

CD_16

CD_23

CD_22

UC_9

UC_14

UC_13

UC_7

UC_10

UC_11

(c)



0

1

2

3

4

5

6

7

8


IBD vs NC

P lt 005


proteins


proteins

Log2 Fold Change

-Log

10P-

valu

e

ITLN1

OLFM4

CD38

CHI3L1

(a)

0

05

1

15

2

25

3

35

4


CD vs UC

P lt 005


proteins


proteins

Log2 Fold Change

-Log

10P-

valu

e

ACE ACE2

(b)

-Log

10 P

val

ue



18

16

14

12

10

8

6

4

2

0

0 001 002 003 004 005

(c)

-Log

10 P

val

ue



8

7

6

5

4

3

2

1

(d)







AOX1

CD38

QPRT

NT5E

NMRK12NMNAT1

PNP

NAPRT

NMRK12

NAMPT

CD38

NADK

NNT

NADSYN1


NAM

NR




NNMT

NADP

NAD

NaADNaMN

QA


NADK2

PNP

NT5C1BNT5C3B

NMNAT2NMNAT3

NMNAT1NMNAT2NMNAT3

NT5C2NT5C NT5C3A

NT5MNT5C1A

NUDT12

ENPP13

NUDT12

ASPDH

Upregulated in IBD


Unchanged

ENPP13

Expression Trends




NAD Degradation

cADPRCD38

NAADPCD38

NAADPR

RYR


NMN



Metabolites

SIRT1NFKB1NFKB2

ADPR

PARP9PARP14

ERSR Ca2+ signaling

Undetected








4 Discussion



0

2

4

6

8

10

GDS3119

NCUCNI

UCI

CD38

relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

lowastlowast

(a)

0

2

4

6

8

GDS2642

NCUCNI

UCI

CD38

relat

ive e

xpre

ssio

n

lowast

lowastns

(b)

GDS3859

0D 2D 4D 6D0

5000

10000

15000

CD38

relat

ive e

xpre

ssio

n

lowast

ns

lowastlowastlowast

(c)

GDS4363

NC 4W 6W

0

5000

10000

15000

20000

CD38

relat

ive e

xpre

ssio

n

lowast

lowast

(d)


















Control UC

CD38

GAPDH

(a)

CD38

GAPDH

Control CD

(b)

3 I 3 NI1 I 1 NI 2 I 2 NI

CD38

GAPDH

(c)

3 NI1 I 1 NI 2 I 2 NI 3 I

CD38

GAPDH

(d)

Control

UC

CD

(e)


Control

UC

CD

(f)




5 Conclusion


Data Availability





Acknowledgments




References























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




























3 Results



TMT15702

TMT25544

TMT35552

4786

329

192 187

287 300

279

(a)

80

60

40

20

0

minus20

minus40

minus60

minus80

t[2]


t[1]NCUCCD

(b)

-3

+3

0

CDUCNC

N_2

N_6

N_3

N_4

N_1

N_5

UC_8

UC_15

UC_12

CD_19

CD_21

CD_20

CD_24

CD_17

CD_18

CD_16

CD_23

CD_22

UC_9

UC_14

UC_13

UC_7

UC_10

UC_11

(c)



0

1

2

3

4

5

6

7

8


IBD vs NC

P lt 005


proteins


proteins

Log2 Fold Change

-Log

10P-

valu

e

ITLN1

OLFM4

CD38

CHI3L1

(a)

0

05

1

15

2

25

3

35

4


CD vs UC

P lt 005


proteins


proteins

Log2 Fold Change

-Log

10P-

valu

e

ACE ACE2

(b)

-Log

10 P

val

ue



18

16

14

12

10

8

6

4

2

0

0 001 002 003 004 005

(c)

-Log

10 P

val

ue



8

7

6

5

4

3

2

1

(d)







AOX1

CD38

QPRT

NT5E

NMRK12NMNAT1

PNP

NAPRT

NMRK12

NAMPT

CD38

NADK

NNT

NADSYN1


NAM

NR




NNMT

NADP

NAD

NaADNaMN

QA


NADK2

PNP

NT5C1BNT5C3B

NMNAT2NMNAT3

NMNAT1NMNAT2NMNAT3

NT5C2NT5C NT5C3A

NT5MNT5C1A

NUDT12

ENPP13

NUDT12

ASPDH

Upregulated in IBD


Unchanged

ENPP13

Expression Trends




NAD Degradation

cADPRCD38

NAADPCD38

NAADPR

RYR


NMN



Metabolites

SIRT1NFKB1NFKB2

ADPR

PARP9PARP14

ERSR Ca2+ signaling

Undetected








4 Discussion



0

2

4

6

8

10

GDS3119

NCUCNI

UCI

CD38

relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

lowastlowast

(a)

0

2

4

6

8

GDS2642

NCUCNI

UCI

CD38

relat

ive e

xpre

ssio

n

lowast

lowastns

(b)

GDS3859

0D 2D 4D 6D0

5000

10000

15000

CD38

relat

ive e

xpre

ssio

n

lowast

ns

lowastlowastlowast

(c)

GDS4363

NC 4W 6W

0

5000

10000

15000

20000

CD38

relat

ive e

xpre

ssio

n

lowast

lowast

(d)


















Control UC

CD38

GAPDH

(a)

CD38

GAPDH

Control CD

(b)

3 I 3 NI1 I 1 NI 2 I 2 NI

CD38

GAPDH

(c)

3 NI1 I 1 NI 2 I 2 NI 3 I

CD38

GAPDH

(d)

Control

UC

CD

(e)


Control

UC

CD

(f)




5 Conclusion


Data Availability





Acknowledgments




References























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















TMT15702

TMT25544

TMT35552

4786

329

192 187

287 300

279

(a)

80

60

40

20

0

minus20

minus40

minus60

minus80

t[2]


t[1]NCUCCD

(b)

-3

+3

0

CDUCNC

N_2

N_6

N_3

N_4

N_1

N_5

UC_8

UC_15

UC_12

CD_19

CD_21

CD_20

CD_24

CD_17

CD_18

CD_16

CD_23

CD_22

UC_9

UC_14

UC_13

UC_7

UC_10

UC_11

(c)



0

1

2

3

4

5

6

7

8


IBD vs NC

P lt 005


proteins


proteins

Log2 Fold Change

-Log

10P-

valu

e

ITLN1

OLFM4

CD38

CHI3L1

(a)

0

05

1

15

2

25

3

35

4


CD vs UC

P lt 005


proteins


proteins

Log2 Fold Change

-Log

10P-

valu

e

ACE ACE2

(b)

-Log

10 P

val

ue



18

16

14

12

10

8

6

4

2

0

0 001 002 003 004 005

(c)

-Log

10 P

val

ue



8

7

6

5

4

3

2

1

(d)







AOX1

CD38

QPRT

NT5E

NMRK12NMNAT1

PNP

NAPRT

NMRK12

NAMPT

CD38

NADK

NNT

NADSYN1


NAM

NR




NNMT

NADP

NAD

NaADNaMN

QA


NADK2

PNP

NT5C1BNT5C3B

NMNAT2NMNAT3

NMNAT1NMNAT2NMNAT3

NT5C2NT5C NT5C3A

NT5MNT5C1A

NUDT12

ENPP13

NUDT12

ASPDH

Upregulated in IBD


Unchanged

ENPP13

Expression Trends




NAD Degradation

cADPRCD38

NAADPCD38

NAADPR

RYR


NMN



Metabolites

SIRT1NFKB1NFKB2

ADPR

PARP9PARP14

ERSR Ca2+ signaling

Undetected








4 Discussion



0

2

4

6

8

10

GDS3119

NCUCNI

UCI

CD38

relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

lowastlowast

(a)

0

2

4

6

8

GDS2642

NCUCNI

UCI

CD38

relat

ive e

xpre

ssio

n

lowast

lowastns

(b)

GDS3859

0D 2D 4D 6D0

5000

10000

15000

CD38

relat

ive e

xpre

ssio

n

lowast

ns

lowastlowastlowast

(c)

GDS4363

NC 4W 6W

0

5000

10000

15000

20000

CD38

relat

ive e

xpre

ssio

n

lowast

lowast

(d)


















Control UC

CD38

GAPDH

(a)

CD38

GAPDH

Control CD

(b)

3 I 3 NI1 I 1 NI 2 I 2 NI

CD38

GAPDH

(c)

3 NI1 I 1 NI 2 I 2 NI 3 I

CD38

GAPDH

(d)

Control

UC

CD

(e)


Control

UC

CD

(f)




5 Conclusion


Data Availability





Acknowledgments




References























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

1

2

3

4

5

6

7

8


IBD vs NC

P lt 005


proteins


proteins

Log2 Fold Change

-Log

10P-

valu

e

ITLN1

OLFM4

CD38

CHI3L1

(a)

0

05

1

15

2

25

3

35

4


CD vs UC

P lt 005


proteins


proteins

Log2 Fold Change

-Log

10P-

valu

e

ACE ACE2

(b)

-Log

10 P

val

ue



18

16

14

12

10

8

6

4

2

0

0 001 002 003 004 005

(c)

-Log

10 P

val

ue



8

7

6

5

4

3

2

1

(d)







AOX1

CD38

QPRT

NT5E

NMRK12NMNAT1

PNP

NAPRT

NMRK12

NAMPT

CD38

NADK

NNT

NADSYN1


NAM

NR




NNMT

NADP

NAD

NaADNaMN

QA


NADK2

PNP

NT5C1BNT5C3B

NMNAT2NMNAT3

NMNAT1NMNAT2NMNAT3

NT5C2NT5C NT5C3A

NT5MNT5C1A

NUDT12

ENPP13

NUDT12

ASPDH

Upregulated in IBD


Unchanged

ENPP13

Expression Trends




NAD Degradation

cADPRCD38

NAADPCD38

NAADPR

RYR


NMN



Metabolites

SIRT1NFKB1NFKB2

ADPR

PARP9PARP14

ERSR Ca2+ signaling

Undetected








4 Discussion



0

2

4

6

8

10

GDS3119

NCUCNI

UCI

CD38

relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

lowastlowast

(a)

0

2

4

6

8

GDS2642

NCUCNI

UCI

CD38

relat

ive e

xpre

ssio

n

lowast

lowastns

(b)

GDS3859

0D 2D 4D 6D0

5000

10000

15000

CD38

relat

ive e

xpre

ssio

n

lowast

ns

lowastlowastlowast

(c)

GDS4363

NC 4W 6W

0

5000

10000

15000

20000

CD38

relat

ive e

xpre

ssio

n

lowast

lowast

(d)


















Control UC

CD38

GAPDH

(a)

CD38

GAPDH

Control CD

(b)

3 I 3 NI1 I 1 NI 2 I 2 NI

CD38

GAPDH

(c)

3 NI1 I 1 NI 2 I 2 NI 3 I

CD38

GAPDH

(d)

Control

UC

CD

(e)


Control

UC

CD

(f)




5 Conclusion


Data Availability





Acknowledgments




References























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















AOX1

CD38

QPRT

NT5E

NMRK12NMNAT1

PNP

NAPRT

NMRK12

NAMPT

CD38

NADK

NNT

NADSYN1


NAM

NR




NNMT

NADP

NAD

NaADNaMN

QA


NADK2

PNP

NT5C1BNT5C3B

NMNAT2NMNAT3

NMNAT1NMNAT2NMNAT3

NT5C2NT5C NT5C3A

NT5MNT5C1A

NUDT12

ENPP13

NUDT12

ASPDH

Upregulated in IBD


Unchanged

ENPP13

Expression Trends




NAD Degradation

cADPRCD38

NAADPCD38

NAADPR

RYR


NMN



Metabolites

SIRT1NFKB1NFKB2

ADPR

PARP9PARP14

ERSR Ca2+ signaling

Undetected








4 Discussion



0

2

4

6

8

10

GDS3119

NCUCNI

UCI

CD38

relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

lowastlowast

(a)

0

2

4

6

8

GDS2642

NCUCNI

UCI

CD38

relat

ive e

xpre

ssio

n

lowast

lowastns

(b)

GDS3859

0D 2D 4D 6D0

5000

10000

15000

CD38

relat

ive e

xpre

ssio

n

lowast

ns

lowastlowastlowast

(c)

GDS4363

NC 4W 6W

0

5000

10000

15000

20000

CD38

relat

ive e

xpre

ssio

n

lowast

lowast

(d)


















Control UC

CD38

GAPDH

(a)

CD38

GAPDH

Control CD

(b)

3 I 3 NI1 I 1 NI 2 I 2 NI

CD38

GAPDH

(c)

3 NI1 I 1 NI 2 I 2 NI 3 I

CD38

GAPDH

(d)

Control

UC

CD

(e)


Control

UC

CD

(f)




5 Conclusion


Data Availability





Acknowledgments




References























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

2

4

6

8

10

GDS3119

NCUCNI

UCI

CD38

relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

lowastlowast

(a)

0

2

4

6

8

GDS2642

NCUCNI

UCI

CD38

relat

ive e

xpre

ssio

n

lowast

lowastns

(b)

GDS3859

0D 2D 4D 6D0

5000

10000

15000

CD38

relat

ive e

xpre

ssio

n

lowast

ns

lowastlowastlowast

(c)

GDS4363

NC 4W 6W

0

5000

10000

15000

20000

CD38

relat

ive e

xpre

ssio

n

lowast

lowast

(d)


















Control UC

CD38

GAPDH

(a)

CD38

GAPDH

Control CD

(b)

3 I 3 NI1 I 1 NI 2 I 2 NI

CD38

GAPDH

(c)

3 NI1 I 1 NI 2 I 2 NI 3 I

CD38

GAPDH

(d)

Control

UC

CD

(e)


Control

UC

CD

(f)




5 Conclusion


Data Availability





Acknowledgments




References























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





























Control UC

CD38

GAPDH

(a)

CD38

GAPDH

Control CD

(b)

3 I 3 NI1 I 1 NI 2 I 2 NI

CD38

GAPDH

(c)

3 NI1 I 1 NI 2 I 2 NI 3 I

CD38

GAPDH

(d)

Control

UC

CD

(e)


Control

UC

CD

(f)




5 Conclusion


Data Availability





Acknowledgments




References























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















Acknowledgments




References























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















Quantitative Proteomic Analysis Reveals the Deregulation of...

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