Antibody-based proteomics for discovery and exploration of proteins expressed in pancreatic islets

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Discovery Medicine, volume 9, number 49, pages 565-578, June 2010

Abstract: Abnormal glucose tolerance and deviantblood glucose levels are late stage clinical parame-ters that signify diabetes mellitus. To be able to diag-nose the disease at an earlier stage and develop newtools for beta cell imaging, new molecular markersare needed. In the present study, five proteins high-ly expressed in pancreatic islets with no expressionin the surrounding exocrine glandular cells of pan-creas, and one protein with the opposite expressionpattern, were identified by searches in the HumanProtein Atlas (www.proteinatlas.org). The proteinswere analyzed immunohistochemically on a special-ly designed tissue microarray, containing isolatedhuman islets and pancreatic tissues with differentcharacteristics, and compared to the expression ofpreviously known markers of endocrine andexocrine pancreatic cells. Of the five novel endocrinemarkers, tetraspanin-7 was identified as a mem-brane-bound protein with exclusive positivity in isletcells. Also β-2-microglobulin and ubiquitin car-boxyl-terminal hydrolase isozyme L1 wereexpressed in a majority of islet cells, whereas

sad1/unc-84 domain-containing protein 1 and beta-1,3-glucuronyltransferase 1 were positive in a small-er subset of islet cells. The potential exocrine mark-er galectin-2 was expressed in both exocrine acinarycells and pancreatic ductal cells, with no or low pos-itivity in islet cells. In conclusion, antibody-basedproteomics and specially designed tissue microar-rays enable identification and exploration of novelproteins with differential expression in pancreaticislets. Here we describe 5 candidate proteins for fur-ther investigation of their physiological role andpotential involvement in the pathogenesis of dia-betes. One of these proteins, tetraspanin-7, isexpressed on the cell membrane and could thus be apotential candidate for future development of trac-ers for beta cell imaging. [Discovery Medicine 9(49):565-

578, June 2010]

Introduction

Diabetes mellitus is diagnosed on the basis of bloodglucose concentration and abnormal glucose tolerancein individuals, a situation not changed over the last 100years. However, these clinical parameters are late stagemarkers for the disease and provide little information asto the changes in pancreatic beta cell function preced-ing the clinical manifestation of the disease.Identification and exploration of novel proteins withdifferential expression in pancreatic islets, and analysisof their physiological role and potential involvement inunderlying mechanisms of beta cell malfunction/destruction, would be of significant clinical advantage.

Antibody-based Proteomics for Discovery

and Exploration of Proteins Expressed

in Pancreatic Islets

CeCilia lindskog, anna asplund, Margareta engkvist, Mathias uhlen,

olle korsgren, and Fredrik ponten

Cecilia Lindskog, M.D., Anna Asplund, M.D., and

Fredrik Ponten, M.D., Ph.D., are at the Department

of Genetics and Pathology, Rudbeck Laboratory,

Uppsala University, Uppsala, Sweden.

Margareta Engkvist, M.D., is at the Department of

Clinical Immunology, Rudbeck Laboratory, Uppsala

University, Uppsala, Sweden.

Mathias Uhlen, M.D., is at the School of

Biotechnology, AlbaNova University Center, Royal

Institute of Technology (KTH), Stockholm, Sweden.

Olle Korsgren, M.D., is at the Department of Clinical

Immunology, Rudbeck Laboratory, Uppsala University,

Uppsala, Sweden.

Discovery Medicine

© Discovery Medicine

http://www.discoverymedicine.com



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Understanding the events that occur in pancreatic betacell mass before the onset of type 1 and type 2 diabetesmellitus could possibly allow early intervention strate-gies to delay or even prevent the onset of disease.

Recent advances in non-invasive imaging technologiessuch as Magnetic Resonance Imaging (MRI) andPositron Emission Tomography (PET) are likely to pro-vide an opportunity to monitor pancreatic beta cell massin humans. A range of candidate proteins and metabo-lites enabling imaging of the islets of Langerhans havereceived significant interest over the past ten years(Schneider, 2008). However, many of these candidateshave lacked the specificity required for in situ imagingof the pancreatic beta cells. The most important issue isthe identification of a beta cell target that is not signifi-cantly expressed in other abdominal tissues or theexocrine pancreas. Furthermore, an essential require-ment for imaging is that the expression level of the pro-tein must be high enough to obtain a good signal-to-noise ratio.

Antibody-based proteomics provides a strategy for thesystematic generation and usage of specific antibodiesto explore the proteome (Uhlen and Ponten, 2005).Based on such a strategy, the Swedish Human ProteinAtlas (HPA) program has been set up to generate a com-prehensive map of protein expression patterns in humantissues and cells (Uhlen et al., 2005). This multi-disci-plinary research program combines large-scale genera-tion of validated antibodies (Nilsson et al., 2005) withprotein profiling in human tissues and cells, using high-throughput immunohistochemistry (Warford et al.,2004) on tissue microarrays (TMAs) (Kononen et al.,1998). The expression of over 8,400 unique proteinscorresponding to 42% of the approximately 20,000 pro-tein encoding genes in the human genome (Clamp et al.,2007) have so far been successfully characterized andpublished on the HPA portal (www.proteinatlas.org)(Berglund et al., 2008). The structure and contents of

this database allow for searches and identification ofproteins expressed in specified tissues (Bjorling et al.,2007), and thus provide an attractive starting point forfurther analysis of identified proteins using other assaysand more targeted patient material.In the present investigation, the HPA portal was used tosearch for proteins with selective expression pattern inpancreas. Based on the immunohistochemical stainingpattern, reliability of the antibody, and previous pub-lished data, six proteins were selected for further evalu-ation in a specially designed TMA containing tissuesfrom isolated human islets exposed to various sub-stances in vitro, and pancreatic tissues from normal anddiabetic subjects.

Materials and Methods

Culturing of pancreatic islets

All human studies were approved by local ethics com-mittees. Once legal consent had been obtained, pancre-atic tissues were procured from multiorgan donorswithin the Nordic Network for Clinical IsletTransplantation. Intraductal enzyme perfusion, auto-mated digestion-filtration, islet continuous gradientpurification, and subsequent islet culture were per-formed as previously described in detail (Goto et al.,2004).

The islets were cultured for 3 days in culturing bagstogether with 100 ml medium (CMRL 1066 AppliChemwith 10% ABO compatible human serum and additives)in four different glucose and cytokine conditions.Detailed descriptions of the culturing conditions arelisted in Table 1.

Tissue microarrays

In vitro cultured pancreatic islets were harvested, fixedin formalin, and dispersed into agarose cell gels for sub-

sequent histoprocessing and paraffinembedding. The paraffin blocks of isletpreparations along with pancreatic tis-sues from both type 2 diabetes mellituspatients and non-diabetic subjects rep-resenting a wide variety of features,were used for production of a TMA.The TMA was generated essentially aspreviously described (Kampf et al.,2004; Andersson et al., 2006), includingthree tissue cores from each donorblock. Descriptions of the characteris-tics of the different pancreatic tissuesare listed in Table 2.

Discovery Medicine, volume 9, number 49, June 2010

proteomics for discovery of proteins expressed in pancreatic islets

Table 1. Culturing Conditions of the In Vitro Islet Preparations

Included in the TMA *

Islets In Vitro -- Culturing Condition Number of Subjects

Low glucose: 2.75 mM 2

Normal glucose: 5.5 mM 2

Normal glucose + cytokines: 5.5 mM + TNF + 50U

IFNγ + 50U IL1β/100ml culturing medium

2

High glucose: 16.7 mM 2

Total 8

* List of in vitro islet preparations exposed to different culturing conditions, includ-ing different glucose levels and addition of cytokines. For each condition, prepara-tions from 2 subjects were included.


https://www.researchgate.net/publication/23139970_A_Genecentric_Human_Protein_Atlas_for_Expression_Profiles_Based_on_Antibodies?el=1_x_8&enrichId=rgreq-260da20f-66c2-49a5-8f9f-a443781693e4&enrichSource=Y292ZXJQYWdlOzQ0ODUxMzQ1O0FTOjEwNDkzNzUwNTY4OTYwM0AxNDAyMDMwNDU5NDk0

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Antibody selection

Antibodies directed towards chromogranin A, insulin,glucagon, and somatostatin were selected for basiccharacterization of subpopulations of endocrine cells inpancreatic islets, and amylase and cytokeratin-19(KRT19) antibodies were used to visualize the exocrinepancreatic cell populations. The antibody recognizingthe pancreas/duodenum homeobox protein 1 (PDX1)was used as a differentiation/maturation marker (Sanderand German, 1997).

Using the advanced search function in the Protein Atlas(Bjorling et al., 2007), 110 protein expression patternswere identified, displaying strong positivity in pancre-atic islet cells, but no expression in exocrine glandularcells of pancreas. The search result is summarized inTable 3. From this list, 5 proteins were selected to beincluded in the present study, based on the immunohis-tochemical staining pattern, reliability of the antibody,and previous published data. Two proteins [sad1/unc-84domain-containing protein 1 (SUNC1) and tetraspanin-7 (TSPAN7)] displayed a cell type specific expressionpattern with positivity essentially restricted to pancreat-ic islet cells. In addition to expression in pancreatic isletcells, 3 proteins [β-2-microglobulin precursor (B2M),ubiquitin carboxyl-terminal hydrolase isozyme L1(UCHL1), and beta-1,3-glucuronyltransferase 1(B3GAT1)] were more ubiquitously expressed in vari-ous tissues, with B3GAT1 being expressed in a subsetof the islet cells.

A search for proteins with an opposite expression pat-tern in pancreas, high expression in exocrine glan-dular cells, and no expression in islet cells identified166 potential exocrine markers. One protein,galectin-2 (LGALS2), was selected for inclusion inthe present study. Detailed information about themanufacturer and dilution of all used primary anti-bodies are summarized in Table 4.

Immunohistochemistry

The TMA sections were immunohistochemicallystained essentially as previously described(Paavilainen et al., 2008). In brief, glass slides werebaked in 60°C for 45 min, deparaffinized in xylene,hydrated in graded alcohols, and blocked forendogenous peroxidase in 0.3% hydrogen peroxide.For antigen retrieval, slides were boiled in TargetRetrieval Solution (Dako, Glostrup, Denmark)using a Decloaking chamber (Biocare Medical,Walnut Creek, CA, USA), then the automatedimmunohistochemistry was performed using anAutoStainer XL ST5010 (Leica Microsystems

GmbH, Wetzlar, Germany). The slides were incubatedfor 30 min at room temperature with primary antibod-ies. For detection, the secondary reagent anti-rabbit/mouse HRP-conjugated UltraVision (ThermoFischer Scientific, Fremont, CA, USA) was used.Following washing steps, the slides were developedwith diaminobenzidine as chromogen. Mayers hema-toxylin (Sigma-Aldrich, St. Louis, MO, USA) was usedas counterstaining. Slides were mounted with Pertex(Histolab AB, Gothenburg, Sweden).

Annotation of immunohistochemically stained

images

The immunohistochemically stained TMA slides werescanned using Aperio ScanScope XT, generating highresolution digital images. The images were used tomanually analyze the patterns of immunoreactivity inislets from both pancreatic tissues and from isolated invitro cultures. The fraction of positive cells for eachantibody and tissue spot was scored using 10%-inter-vals.

Western blotting

To further validate the antibodies, the 7 antibodies tar-geting proteins previously characterized in pancreaswere analyzed with Western blotting using 4 lysatesfrom human pancreatic tissues, including 2 preparationsof isolated pancreatic islets and 2 exocrine pancreatictissues, of which 1 islet lysate and 1 exocrine lysatewere treated with protease inhibitor. The 6 less knownantibodies were tested using 5 lysates -- 2 preparationsof isolated pancreatic islets, 2 exocrine pancreatic tis-

Table 2. Characteristics of the Pancreatic Tissues

Included in the TMA *

Islets In Vitro -- Characteristics Number of Subjects

Type 2 diabetes mellitus 2

High BMI: > 40 2

Low BMI: < 20 2

High age: > 75 years 2

Low age: < 25 years 2

Long ischemia: > 22 h 2

Short ischemia: < 4 h 2

Low stimulation index: <2 2

Medium stimulation index: 2-15 2

High stimulation index: >15 2

* List of whole pancreatic tissues selected from type 2 diabetespatients and from subjects with different BMI, age, time of ischemia,and stimulation index. For each group, tissues from two subjects wereincluded.


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Table 3 (Part 1). Culturing Conditions of the In Vitro Islet Preparations Included in the TMA (Part 1) *

# Gene Name Description Antibody ID

1 ABCF2 ATP-binding cassette sub-family F member 2 CAB020682

2 AC008898.6 UPF0514 membrane protein FAM159B HPA011778

3 ACLY ATP-citrate synthase HPA022959

4 ACOX1 Peroxisomal acyl-coenzyme A oxidase 1 HPA021195

5 ADNP2 ADNP homeobox protein 2 HPA007126

6 AL136139.6 HERV-FRD_6p24.1 provirus ancestral Env polyprotein HPA011812

7 AMPH Amphiphysin HPA019829

8 ANGPT4 Angiopoietin-4 Precursor CAB013260

9 ANXA7 Annexin A7 CAB004312

10 ARG2 Arginase-2, mitochondrial Precursor CAB009435

11 ASB9 Ankyrin repeat and SOCS box protein 9 HPA003014

12 ATP6AP2 Renin receptor HPA003156

13 B2M Beta-2-microglobulin Precursor HPA006361

14 B3GAT1 Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1 CAB002500

15 BTG1 Protein BTG1 HPA005972

16 C4ORF18 Uncharacterized protein C4orf18 HPA007227

17 C5ORF40 Fibronectin type-III domain-containing protein C5orf40 HPA017291

18 CCDC116 Coiled-coil domain-containing protein 116 HPA000853

19 CCND3 G1/S-specific cyclin-D3 CAB000116

20 CD99 CD99 antigen Precursor CAB000020

21 CDH2 Cadherin-2 Precursor CAB000141

22 CHGA Chromogranin-A CAB000023 / HPA017369

23 CHGB Secretogranin-1 Precursor HPA008759 / HPA012602

24 CHRM1 Muscarinic acetylcholine receptor M1 HPA014101

25 CLU Clusterin HPA000572

26 CPE Carboxypeptidase E Precursor CAB024907

27 CYP2W1 Cytochrome P450 2W1 HPA012753

28 DACH2 Dachshund homolog 2 HPA000258

29 DFFB DNA fragmentation factor subunit beta CAB004328

30 DGCR2 Integral membrane protein DGCR2/IDD Precursor HPA000873

31 ELMOD3 ELMO domain-containing protein 3 HPA012126

32 ENO3 Beta-enolase HPA000793

33 F9 Coagulation factor IX HPA000254

34 FAM187B Protein FAM187B Precursor HPA014687

35 FRS2 Fibroblast growth factor receptor substrate 2 CAB010347

36 FTHL16 Ferritin heavy chain CAB008623

37 GC Vitamin D-binding protein Precursor HPA019855

38 GCG Glucagon CAB000040

39 GDA Guanine deaminase HPA019352

40 GLP2R Glucagon-like peptide 2 receptor Precursor CAB022690

41 GMPR GMP reductase 1 HPA021476

42 GNAS Neuroendocrine secretory protein 55 HPA018122

43 GPR44 Putative G-protein coupled receptor 44 HPA014259

44 HEATR4 HEAT repeat-containing protein 4 HPA003642

45 HLA-DQB1 HLA class II histocompatibility antigen, DQB1*0602 beta chain Precursor HPA013667

46 HMGXB3 Protein SMF HPA002354

47 HSPA12A Heat shock 70 kDa protein 12A HPA011273

48 IAPP Islet amyloid polypeptide Precursor CAB000352

49 IGFBP7 Insulin-like growth factor-binding protein 7 Precursor CAB020668

50 IL10 Interleukin-10 Precursor CAB013120

51 IL8RB High affinity interleukin-8 receptor B CAB016268

52 INHBA Inhibin beta A chain Precursor HPA020031

53 INS Insulin Precursor CAB000048 / HPA004932 / CAB012098

54 KBTBD10 Kelch repeat and BTB domain-containing protein 10 HPA021753

55 KIAA0323 Protein KIAA0323 HPA003402

* List of in vitro islet preparations exposed to different culturing conditions, including different glucose levels and addition of cytokines. For each condi-tion, preparations from 2 subjects were included.


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Table 3 (Part 2). Culturing Conditions of the In Vitro Islet Preparations Included in the TMA *

# Gene Name Description Antibody ID

56 KIF3B Kinesin-like protein KIF3B HPA007119

57 LEP Leptin Precursor CAB016730

58 MAGEB17 Putative MAGE domain-containing protein HPA003756

59 MAP2 Microtubule-associated protein 2 HPA008273

60 MCTP1 Multiple C2 and transmembrane domain-containing protein 1 HPA019018

61 MID1 Midline-1 HPA003715

62 MPDZ Multiple PDZ domain protein HPA020255

63 NCAM1 Neural cell adhesion molecule 1 Precursor CAB000142

64 NPDC1 Neural proliferation differentiation and control protein 1 Precursor HPA008189

65 NPSR1 Neuropeptide S receptor HPA007489

66 NUCB1 Nucleobindin-1 Precursor HPA008176

67 P2RY4 P2Y purinoceptor 4 CAB022644

68 PAFAH2 Platelet-activating factor acetylhydrolase 2, cytoplasmic HPA018157

69 PBXIP1 Pre-B-cell leukemia transcription factor-interacting protein 1 HPA006949

70 PCP4 Purkinje cell protein 4 HPA005792

71 PDLIM1 PDZ and LIM domain protein 1 HPA017010

72 PLEK2 Pleckstrin-2 HPA001208

73 POLG2 DNA polymerase subunit gamma-2, mitochondrial Precursor HPA023202

74 POLI DNA polymerase iota HPA012000

75 PPY Pancreatic prohormone Precursor CAB000069

76 PRSSL1 Serine protease 1-like protein 1 Precursor HPA006099

77 PTPRN Receptor-type tyrosine-protein phosphatase-like N Precursor HPA007179

78 RAB3B Ras-related protein Rab-3B HPA003159 / CAB023293

79 RAB5C Ras-related protein Rab-5C HPA003426

80 RBP4 Plasma retinol-binding protein HPA001641 / CAB004555

81 RTN1 Reticulon-1 CAB002745

82 SCG3 Secretogranin-3 Precursor HPA006880

83 SCG5 Neuroendocrine protein 7B2 HPA013136

84 SCGN Secretagogin CAB004005 / HPA006641

85 SERPINB2 Plasminogen activator inhibitor 2 Precursor HPA015480

86 SH3GL2 Endophilin-A1 CAB010056

87 SNAP25 Synaptosomal-associated protein 25 HPA001830

88 SNCB Beta-synuclein CAB002681

89 SST Somatostatin Precursor HPA019472

90 STRADB STE20-related kinase adapter protein beta HPA026549

91 STX1A Syntaxin-1A CAB008372

92 STXBP1 Syntaxin-binding protein 1 HPA023483

93 SUNC1 Sad1/unc-84 domain-containing protein 1 HPA008344

94 SV2A Synaptic vesicle glycoprotein 2A CAB002226 / HPA007863

95 SYP Synaptophysin CAB000076

96 TFF3 Trefoil factor 3 Precursor CAB020681

97 TMED8 Protein TMED8 HPA001205

98 TMEM184B Transmembrane protein 184B HPA024076

99 TNFRSF21 Tumor necrosis factor receptor superfamily member 21 Precursor CAB009805

100 TOM1L2 TOM1-like protein 2 HPA022541

101 TRPV4 Transient receptor potential cation channel subfamily V member 4 HPA007150

102 TSPAN3 Tetraspanin-3 HPA015996

103 TSPAN7 Tetraspanin-7 HPA003140

104 TTR Transthyretin Precursor HPA002550

105 TXNRD1 Thioredoxin reductase 1, cytoplasmic HPA001395

106 UCHL1 Ubiquitin carboxyl-terminal hydrolase isozyme L1 CAB002580 / HPA005993

107 WDR31 WD repeat-containing protein 31 HPA019340

108 ZCCHC6 Zinc finger CCHC domain-containing protein 6 HPA020615

109 ZNF192 Zinc finger protein 192 HPA003483

110 ZSWIM5 Zinc finger SWIM domain-containing protein 5 HPA018211

* List of in vitro islet preparations exposed to different culturing conditions, including different glucose levels and addition of cytokines. For each condi-tion, preparations from 2 subjects were included.


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sues, and 1 whole pancreatic tissue.

Statistical analysis

Statistical evaluation was performed using theStatistical Package for the Social Sciences (SPSS,Chicago, IL, USA). Results are expressed as mean ±standard deviation. Unpaired t tests were used to test forbetween-group differences in fraction of positive cells,with p < 0.05 being considered significant.

Results

An in silico discovery strategy based on the advancedsearch tool (Bjorling et al., 2007) of the HPA portal wasused to find proteins with selective expression in theendocrine cells of human pancreas. A search for pro-teins that are highly expressed in pancreatic islets butnot expressed in the exocrine parenchyma resulted in alist of 110 proteins, including proteins with previouslywell-known functions as well as unknown proteins(Table 3). Five proteins -- B3GAT1, B2M, SUNC1,TSPAN7, and UCHL1 -- not previously characterized inpancreatic islet cells, as well as LGALS2, a potential

marker for exocrine glandular cells, were selected formore in-depth protein profiling.

Basic characterization of pancreatic islets

A specially designed TMA was generated includingpancreatic tissues from healthy individuals and patientswith type 2 diabetes mellitus, as well as isolated andcultured pancreatic islets exposed to various concentra-tions of glucose and cytokines in vitro. Using immuno-histochemistry, the expression pattern of chromograninA, insulin, glucagon, somatostatin, and PDX1 wasdetermined in pancreatic tissues and isolated isletpreparations (Figure 1) by manual microscopic evalua-tion.

For chromogranin A, insulin, and somatostatin, distinctcytoplasmic immunoreactivity with a homogenous pat-tern was observed both in islets in pancreatic tissues andin in vitro cultured islets. The staining pattern ofglucagon was heterogeneous and more diffuse in the invitro cultured islets compared to pancreatic tissue islets,with a larger fraction of the cells displaying weak tomoderate cytoplasmic immunoreactivity, in addition to

the subset of cells with strongimmunoreactivity. PDX1 wasstrongly stained in nuclei of pancre-atic islets, but to a lesser extent alsoin nuclei of exocrine ductal cells.Mean values of positive cells ± stan-dard deviation were comparedbetween islets in pancreatic tissuesand in vitro cultured islets. Areduced fraction of positive cells inin vitro cultured islets compared toislets in pancreatic tissues wasobserved for chromogranin A (100 ±0.0% vs. 80 ± 19.1%; p < 0,001),insulin (85 ± 8.8% vs. 46 ± 11.9%; p< 0.001), and PDX1 (78 ± 22.6% vs.36 ± 13.4%; p < 0.001), whereasthere was no significant differencein the amount of glucagon (11 ±11.8% vs. 15 ± 7.4%; p = 0.45) andsomatostatin (9 ± 5.2% vs. 9 ±2.5%; p = 1.0) expressing cells.

Of the exocrine markers (Figure 2),amylase was found to be distinctlypositive in exocrine glandular cellsof pancreatic tissues. In 8 ± 5.0% ofthe in vitro cultured islets, strongcytoplasmic immunoreactivity witha granular or dot-like pattern wasobserved. LGALS2 was strongly

Table 4. Antibodies Included in the Study *

Protein Name Antibody ID Clone Manufacturer Dilution

Chromogranin-A CAB000023 LK2H10 BoehringerMannheim

1/5000

Insulin HPA004932 - Atlas Antibodies 1/1000

Glucagon CAB000040 pAb Dako Cytomation 1/1000

Somatostatin CAB000075 pAb Dako Cytomation 1/4000

Pancreas/duodenumhomeobox protein 1

CAB025873 267712 R&D Systems 1/50

Alpha-amylase 1 CAB004310 G-10 Santa CruzBiotechnology

1/20000

Galectin-2 HPA003536 - Atlas Antibodies 1/50

Keratin, type Icytoskeletal 19

CAB000031 RCL108 Dako Cytomation 1/50

Beta-2-microglobulinPrecursor

HPA006361 - Atlas Antibodies 1/250

Beta-1,3-glucuronyl-transferase 1

CAB002500 NK-1 Novocastra 1/1

Sad1/unc-84 domain-containing protein 1


Tetraspanin-7 HPA003140 - Atlas Antibodies 1/100

Ubiquitin carboxyl-terminal hydrolaseisozyme L1


* List of antibodies used in the present study, with details about their manufacturer andantibody dilution.


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Figure 1. Mean values of the fraction of positive

islet cells (a), and the immunohistochemical

staining pattern (b) in situ and in vitro for chro-

mogranin A, insulin, glucagon, somatostatin, and

PDX1.

(a) A reduced fraction of positive cells in in vitro

cultured islets (right bars) compared to islets in

situ (left bars) was observed for chromogranin A,

insulin, and PDX1 (p < 0.001). No difference was

seen for glucagon (p = 0.45) and somatostatin (p

= 1.00).

(b) Chromogranin A (top), insulin (second from

top), and somatostatin (second from bottom) dis-

played strong cytoplasmic positivity with a

homogenous pattern in both islets in situ (left)

and in vitro cultured islets (right). For glucagon

(middle) a distinct cytoplasmic immunoreactivity

was observed in islets in situ; however, the posi-

tivity in in vitro cultured islets was heterogeneous

and more diffuse, showing weak to moderate

cytoplasmic staining in a majority of the islet

cells and strong positivity in a smaller fraction of

the islet cells. PDX1 (bottom) displayed strong

nuclear immunoreactivity both in islets in situ and

in islets in vitro. Furthermore, some cells in

exocrine glandular cells were positive (bottom

left).

* = p < 0.05


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expressed in cytoplasm and nuclei in a majority ofexocrine glandular cells. In some cells a polarity wasobserved, and few cells were more weakly stained. Inthe in vitro cultured islets, 11 ± 2.4% of the cells dis-played strong expression of LGALS2. KRT19 exhibit-ed distinct cytoplasmic and membranous immunoreac-tivity in ductal cells of pancreatic tissues and in 15 ±

5.3% of the in vitro cultured islets. All remaining cellswere negative.

Expression pattern of selected proteins

All five novel endocrine markers showed expression inislet cells, both in pancreatic tissues and in in vitro cul-tured islets (Figure 3). B2M, a secreted protein part of

Figure 2. Mean values of the frac-

tion of positive islet cells (a), and

the immunohistochemical staining

pattern (b) in situ and in vitro for

amylase, LGALS2, and KRT19.

(a) All three exocrine markers were

positive in ≤15 % of the cells in in

vitro cultured islets. The fraction of

positive islet cells in situ was not

evaluated.

(b) Amylase (top) displayed strong

cytoplasmic positivity in exocrine

glandular cells, but negative in islets

in situ (left). In in vitro cultured islet

(right), strong immunoreactivity

with a granular cytoplasmic pattern

was observed in a fraction of the

cells. LGALS2 (middle) was strong-

ly stained in cytoplasm and nuclei of

exocrine glandular cells, whereas

islets in situ were negative. Some

exocrine cells had a polarity in the

staining pattern. In islets in vitro a

small amount of the cells displayed

strong cytoplasmic and nuclear

immunoreactivity. KRT19 (bottom)

displayed strong cytoplasmic stain-

ing with a membranous pattern in

exocrine ductal cells; islets in situ

were negative. A fraction of the cells

in in vitro cultured islets were posi-

tive in a similar pattern as the ductal

cells.

† not evaluated


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Figure 3. Mean values of the fraction of positive

islet cells (a), and the immunohistochemical

staining pattern (b) in situ and in vitro for B2M,

B3GAT1, SUNC1, TSPAN7, and UCHL1.

(a) A reduced fraction of positive cells in in vitro

cultured islets (right bars) compared to islets in

situ (left bars) was observed for B3GAT1 (p =

0.02), SUNC1 (p < 0.001), TSPAN7 (p < 0.001),

and UCHL1 (p < 0.001). No significant differ-

ence was seen for B2M (p = 0.18).

(b) B2M (top) displayed moderate to strong cyto-

plasmic and partly membranous staining in a

majority of islets in situ (left) and in vitro (right),

often stronger in areas close to the plasma mem-

brane. B3GAT1 (second from top) was strongly

positive in cytoplasm, with a granular or dot-like

pattern both in islets in situ and in vitro. SUNC1

(middle) displayed strong cytoplasmic positivity

in islets in situ as well as in islets in vitro. The

staining pattern was often granular, and in the in

vitro cultured islets often accentuated to a smaller

area of the cytoplasm. TSPAN7 (second from bot-

tom) displayed distinct cytoplasmic immunoreac-

tivity both in islets in situ and islets in vitro, often

with a polarity and with intensities varying from

weak to strong immunoreactivity. UCHL1 (bot-

tom) was strongly expressed in cytoplasm and

nuclei in in vitro cultured islets. The islets in situ

displayed a similar pattern, however, of a slightly

weaker intensity.

* = p < 0.05


major histocompatibility complex class I molecules,was strongly expressed in 88 ± 8.0% of the islet cells inpancreatic tissues, compared to 83 ± 14.6% (p = 0.18)in the in vitro cultured islets, with stronger immunore-activity being observed close to the plasma membrane.The number of B2M expressing cells resembled that ofchromogranin A expressing ones. B3GAT1 is involvedin glycoprotein biosynthesis and displayed distinctcytoplasmic immunoreactivity with a granular or dot-like pattern in only a small fraction of the islet cells,most similar to the pattern of somatostatin expressingcells. In islets of pancreatic tissues, 12 ± 7.4% of thecells were positive, while the fraction of positive cellswas significantly smaller (5 ± 2.5%; p = 0.02) in in vitrocultured islets. Another protein with highly selectiveexpression in Langerhans islets was SUNC1, a proteinwith unknown function. SUNC1 displayed a partlygranular cytoplasmic expression pattern in a majority ofthe pancreatic islet cells. The distribution resembledthat of insulin in islets of the pancreatic tissues, withpositivity in 69 ± 17.0% of the cells. The amount ofpositive cells was significantly lower in the in vitro cul-tured islets (14 ± 4.9%; p < 0.001). TSPAN7 is a trans-membrane protein suggested to be involved in cell dif-ferentiation (Boismenu et al., 1996) and cell motility(Penas et al., 2000), displaying distinct cytoplasmicimmunoreactivity in islet cells with a distribution simi-lar to insulin. The intensity of the TSPAN7 immunos-taining was variable in islet cells, ranging from weak tostrong, with 85 ± 17.0% of the islet cells in pancreatictissues being positive. A reduced fraction of positivecells was observed in in vitro cultured islets (51 ±19.9%; p < 0.001). UCHL1, a cytoplasmic proteininvolved in regulation of protein degradation, wasstrongly expressed in a majority of cells in pancreaticislets (80 ± 15.0%) and to a lesser extent in in vitro cul-tured islets (44 ± 9.2%; p < 0.001). The cytoplasmicstaining pattern was homogenous and often accompa-nied with nuclear immunoreactivity.

Protein expression in different groups of patients

For most of the proteins investigated, there was no dif-ference in the level of expressed protein in islets sub-jected to different culture conditions or in islets of pan-creatic tissues from the different groups of patients.Although not significant, some trends were noteddespite the few samples representing each group. Isletpreparations cultured in high concentration of glucose(16.7 mM) had lower expression of PDX1 and B2M,with 57 ± 23.6% (PDX1) and 63 ± 14.1% (B2M) posi-tive islet cells in high glucose compared to 78 ± 3.5%(PDX1) and 82 ± 2.4% (B2M) in normal glucose. In invitro cultured islets grown in normal glucose concentra-tion (5.5mM) with supplemented additional cytokines

(50U TNF + 50U Interferon γ + 50U IL1β/100ml cul-turing medium), a higher expression of B2M andUCHL was found. 98 ± 2.4% (B2M) and 47 ± 9.4%(UCHL) of islet cells were positive as compared to 82± 2.4% (B2M) and 38 ± 2.4% (UCHL) positive isletcells in islets cultured without the supplement ofcytokines. In islets from pancreatic tissues, the expres-sion of B2M and TSPAN7 was reduced in tissues sub-jected to long time of ischemia (>22 h) as compared toshort time of ischemia (<4 h). Prolonged ischemiaresulted in 80 ± 9.4% (B2M) and 63 ± 24.8%(TSPAN7) positive islet cells as compared to 97 ± 0.0%(B2M) and 98 ± 2.4% (TSPAN7) in tissues with onlyshort duration of ischemia. There was no obvious ten-dency towards a difference in protein expression levelsbetween type 2 diabetes mellitus patients and the otherdonators for any of the analyzed proteins.

Western blotting

To further validate the antibodies used in the study andto examine if the protein expression patterns found inimmunohistochemistry also were reflected in Westernblot, all antibodies were analyzed with Western blot-ting, using lysates from isolated endocrine- andexocrine pancreatic cells (Figure 4). Of the 7 proteinspreviously well-known to be expressed in pancreas,bands of predicted size were found for insulin, somato-statin, amylase, and KRT19. Chromogranin A displayedbands of predicted size, but also several additionalbands of non-predicted sizes. Glucagon and PDX1 dis-played larger bands than the predicted sizes, which forPDX1 was expected according to previous results (Li etal., 2010). The addition of protease inhibitor to the tis-sues did not result in any difference for any of the anti-bodies (Figure 4a).

Of the antibodies corresponding to the 6 newly identi-fied proteins with differential expression in pancreas,B2M, UCHL1, and LGALS2 detected proteins of pre-dicted size, with UCHL1 exhibiting strong bands inlysates from islet cells and LGALS2 displaying strongbands in lysates from exocrine cells. B2M showedbands of predicted size both in wells representing isletcells and in wells representing exocrine cells. SUNC1displayed distinct bands smaller than predicted inlysates from islet cells and in one of the two lysatesfrom exocrine cells. TSPAN7 exhibited strong bands inclose vicinity to the loading well, only in lysates fromislet cells. No bands were seen for B3GAT1.

The discrepancies between immunohistochemistry andWestern blotting for certain antibody-protein interac-tions are not unexpected, as protein epitopes are alteredin different ways dependent on effects of denaturing,

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e.g., formalin and SDS-PAGE. The reason for TSPAN7to be remaining near the loading wells is unclear andmay be due to complex binding or modifications of theTSPAN7 protein. Dilution of the lysate did not alter theability of TSPAN7 to move further into the gel.

Discussion

The identification of specific gene expression patternsis a major challenge to increase our understanding ofnormal islet function and the pathogenesis of diabetes.The discovery of new proteins with a selective expres-sion in Langerhans islets as compared to surrounding

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Figure 4. Western blot results of the

markers selected for basic characteriza-

tion of pancreatic islets (a) and of the

selected proteins previously not charac-

terized in pancreas (b).

(a) Insulin (II) and somatostatin (IV)

had bands of predicted size in the lanes

representing lysates from pancreatic

islets (isl), whereas the lanes with

lysates from exocrine tissue (exo) dis-

played weaker bands. Amylase (V) and

KRT19 (VI) recognized proteins of pre-

dicted sizes in exocrine tissue, while the

bands of pancreatic islets were weaker.

For chromogranin A (I), several bands

were seen, of both expected size and of

too large or too small sizes, mainly with

lysates from pancreatic islets but also

with lysates from exocrine tissue.

Glucagon (III) and PDX1 (VII) dis-

played distinct bands of too large size.

For glucagon, bands were presented in

lanes representing both lysates from

pancreatic islets and exocrine tissue,

whereas for PDX1, bands were exclu-

sively found for lysates in pancreatic

islets. For none of the antibodies, no dif-

ference was observed after adding pro-

tease inhibitor (PI) to the lysates.

(b) B2M (I), UCHL1 (V), and LGALS2

(VI) recognized proteins of predicted

sizes. For B2M, strongest bands were

observed in the lanes for exocrine tissue

(exo), whereas the lanes for pancreatic

islets (isl) displayed weaker bands. No

bands were seen in the lane representing

lysate from whole pancreatic tissue

(pancreas). UCHL1 recognized pro-

teins exclusively in lanes for pancreatic

islets, and LGALS2 displayed bands

only in the lanes for exocrine tissue and

whole pancreatic tissue. For B3GAT1

(II) no bands were seen, whereas for

SUNC1 (III), bands of too small size

were observed, mainly in the lanes rep-

resenting pancreatic islets, but also in

one lane for exocrine tissue. The bands

for TSPAN7 (IV) were found in close

vicinity to the loading well, only in the

lanes representing pancreatic islets.


exocrine pancreas also provides a starting point for theidentification and development of candidates to deter-mine beta cell mass. Tissue-restricted transcripts havebeen characterized by various groups, and efforts usingcDNA libraries have established a relative abundanceof >2,000 islet transcripts, including both well-knownand potential new markers (Cras-Meneur et al., 2004).Alternative efforts using oligonucleotide chips havealso been employed to uncover markers of healthy ordiseased islet cell masses (Maffei et al., 2004).Furthermore, genes of pancreatic islets modified byviral infections and cytokines have been described(Ylipaasto et al., 2005). In order to understand the cor-relation between genotype and phenotype, an importantcomplement to transcript profiling is to determine pro-tein profiles in a tissue context. Two-dimensional gelelectrophoresis and mass spectrometry have been usedto generate reference maps of the human pancreaticislet proteins or peptides as a resource for future analy-ses of pancreatic islet biology (Ahmed et al., 2005;Metz et al., 2006). Further development of mass spec-trometry-based technologies to overcome sensitivitylimitations will most probably become increasinglyimportant in the search for proteins involved in beta cellbiology.

An alternative proteomics approach is antibody-basedproteomics (Uhlen and Ponten, 2005), which relies onimmunohistochemistry and prevails as an invaluablemethod for in situ visualization of protein expressionpatterns. Although immunohistochemistry is not aquantitative method, it allows for the detection andlocalization of defined proteins in a tissue context atcellular or subcellular resolution, provided that protein-specific antibodies are available. The HPA program hasemployed a strategy to generate antibodies towardshuman proteins on a global scale and to use these to cre-ate a comprehensive atlas of protein expression patternsin human normal and cancer tissues as well as in celllines. In addition to protein profiling using immunohis-tochemistry and brightfield microscopy, confocalmicroscopy with fluorescently labeled antibodies isalso used to provide a more detailed analysis and high-er resolution of the subcellular localization pattern ofeach protein (Barbe et al., 2008). This resource withover 9 million annotated images can be used to identifyproteins with cell- and tissue type specific expressionpatterns, and to detect various types of biomarkersusing in silico based methods (Bjorling et al., 2007). Alarge fraction of the human genome is expressed on theprotein level in any given cell type and protein expres-sion restricted to only a single cell type is very uncom-mon (Ponten et al., 2009). However, when searching forproteins expressed in a certain cell type as compared toother surrounding cell types within a defined organ,

e.g., Langerhans islet cells and exocrine epithelial cellsin the pancreas, there are substantially more proteinsthat meet the requirement of being differentiallyexpressed.

The general outline of immunohistochemistry on TMAsis well suited for more targeted studies, and with ade-quate materials available, any tissue or disease can bestudied in extended and more in-depth analyses (Pontenet al., 2008). The use of TMAs, containing tissues rep-resenting large patient cohorts coupled to clinical data-bases, also allows for controlled studies on single slidessparing valuable tissues and using only small amountsof reagent (Kononen et al., 1998). Since all tissues areprocessed and analyzed simultaneously, experimentalvariance is minimized, rendering interpretation ofresults more accurate and robust. In the present studywe have used an experimental set-up combining queriesin the HPA database with immunohistochemistry andTMA technology, applied on pancreatic islet cells toaddress questions regarding diabetes and islet cell biol-ogy. Based on the immunohistochemical staining pat-tern, antibody reliability, and previous published data,six proteins distinctly expressed in defined cells of pan-creatic tissue were selected for this study. The aim wasto determine the expression pattern of the selected pro-teins in in vitro cultured islet cell preparations exposedto different conditions, and on pancreatic tissues fromsubjects representing different states of the metabolicsyndrome.

The protein expression pattern of B2M and UCHL1,with cytoplasmic positivity in a majority of the isletcells, is consistent with expression corresponding to thebeta cells. To establish exclusive expression of theseproteins in beta cells, further studies are needed usingdouble labeling techniques and other in vitro assays.Previous studies have revealed that major histocompat-ibility complex class I molecules, which B2M is part of,are hyperexpressed by the endocrine cells during thepathogenic process of type 1 diabetes mellitus (Foulis,1996), and B2M is produced at high levels in isletsfrom post-mortem pancreatic tissue in an individualwho repeatedly tested positive for islet cell antibodies(Oikarinen et al., 2008). In addition, it is suggested thatan intact B2M-pathway is necessary for islet allograftsurvival in mice (Beilke et al., 2004). Although not sig-nificant, the expected physiological processes werereflected when comparing the protein expression inpancreatic islets with different characteristics, e.g.,addition of cytokines resulted in altered expression lev-els of B2M. TSPAN7 was expressed to a similar extentas B2M and UCHL1, although this protein appearedwith a hinted polarity in positive islet cells. Providedthat the expression of TSPAN7 is exclusively expressed

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in pancreatic islets and that the protein, which containstransmembrane spanning regions, also is expressed onthe cell surface, TSPAN7 may be a potential candidatefor future development of tracers for beta cell imaging.LGALS2 displayed a distinct immunohistochemicalstaining pattern restricted to exocrine cells of pancreasas compared to islets. The LGALS2 staining patternappeared less diffuse than what was found using anantibody towards amylase, suggesting that LGALS2could be used as a marker for exocrine glandular cells,independent of the level of exocrine granules within thecells. In general, antibodies recognizing proteins withselective positivity in islet cells displayed a reducedamount of positive islet cells in the in vitro culturedislets compared to the islets in pancreatic tissues. Thisresult is consistent with previous findings of slightimpurity of the islet isolations, due to a remaining con-tribution of exocrine and ductal cells in the cell culture(Shapiro et al., 2000). This was also confirmed withbasic characterization of the islet cells with antibodiesagainst amylase, LGALS2, and KRT19.

Conclusions

In conclusion, our study describes an efficient strategyto identify novel protein targets that are specificallyexpressed in pancreatic islet cells, and gives an exam-ple of how the identified targets can be further exploit-ed in a selected set of well-defined tissues and in vitrocultured islets subjected to experimental perturbations.Larger cohorts, deeper characterization, and functionalstudies are needed to transform the discovery and iden-tification of proteins with islet cell specific expressionpatterns and to further understand islet cell biology andthe development of new candidates for clinical beta cellimaging.

Acknowledgments

The authors wish to acknowledge all members of theHuman Protein Atlas project and the Department ofClinical Immunology, Uppsala University for makingthis work possible. The project is financially supportedby The Wallenberg Research Foundation (KAW), theSwedish Research Council (VR), the Juvenile DiabetesResearch Foundation, and Vinnova Proj# 30552-1.

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Antibody-based proteomics for discovery and exploration of proteins expressed in pancreatic islets

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