Open Access

Identification of Glycoproteins from Mouse Skin Tumors and Plasma

  • Yuan Tian1,
  • Karen S. Kelly-Spratt2,
  • Christopher J. Kemp2 and
  • Hui Zhang3Email author
Clinical Proteomics20084:9014

DOI: 10.1007/s12014-008-9014-z

Received: 12 February 2008

Accepted: 21 July 2008

Published: 4 September 2008

Abstract

Introduction

Plasma has been the focus of testing different proteomic technologies for the identification of biomarkers due to its ready accessibility. However, it is not clear if direct proteomic analysis of plasma can be used to discover new marker proteins from tumors that are associated with tumor progression. In this paper, we reported that such proteins can be detected in plasma in a chemical-induced skin cancer model in mice.

Materials and Methods

We analyzed glycoproteins from both benign papillomas and malignant carcinomas from mice using our recently developed platform, solid-phase extraction of glycopeptides and mass spectrometry, and identified 463 unique N-linked glycosites from 318 unique glycoproteins. These include most known extracellular proteins that have been reported to play roles in skin cancer development such as thrombospondin, cathepsins, epidermal growth factor receptor, cell adhesion molecules, cadherins, integrins, tuberin, fibulin, and TGFβ receptor. We further investigated whether these tumor proteins could be detected in plasma from tumor-bearing mice using isotope labeling and 2D liquid chromatography/matrix-assisted laser desorption/ionization tandem mass spectrometry.

Results and Discussion

Two tumor glycoproteins, Tenascin-C and Arylsulfatase B, were identified and quantified successfully in plasma from tumor bearing mice. This result indicates that analysis of tumor-associated proteins in tumors and plasma by a method using glycopeptide capture, isotopic labeling, and mass spectrometry can be used as a discovery tool to identify candidate tumor proteins that may be detected in plasma.

Keywords

Cancer Plasma Tumor Glycoprotein Proteomics Mass spectrometry MALDI-TOF/TOF MS/MS Serum Plasma Biomarker

Introduction

Despite the great increase in understanding of cancer at the molecular level, cancer remains as the second most common cause of death in the USA. Survival rates for many common cancer types have changed little over the past two decades [1]. If cancer is detected early, prior to metastatic spread, survival rates are vastly improved [1]. For this reason, improvements in the ability to detect cancer early may significantly reduce mortality from cancer. Plasma has been the focus for applying different proteomic technologies for the identification of biomarkers for early detection due to its ready accessibility. These developments include depletion of the most abundant plasma proteins [2] and extensive fractionation of proteins or peptides prior to mass spectrometric analysis [35]. However, proteins discovered by serum profiling are often well-known, high-abundance, classical serum proteins [6], not likely to be specifically derived from cancer tissue. Useful biomarkers for cancer detection in blood are those proteins released specifically from cancer tissues (overexpression of cancer proteins), indicators of a specific response of the host to cancer cells, or leaking of organ restricted proteins to blood due to structural changes in the microenvironment surrounding cancer cells (leaking of normal proteins such as PSA) [7]. Tumor proteins that are detectable in both benign and malignant tumors, as well as plasma can serve as candidate proteins for early detection of cancer. Detection of these proteins in plasma is critical to evaluate proteomic technologies for the biomarker discovery.

In an attempt to identify the proteins derived from cancerous tissue that are most likely to be present in blood, we employed our recently developed glycoproteomic analysis method using solid-phase extraction of N-linked glycopeptides (SPEG) [810]. The method has several advantages. First, most cell-surface and secreted proteins are glycosylated, and disease-associated glycoproteins (secreted by cells, shed from their surface, or otherwise released) are likely to enter the bloodstream and thus represent a rich source of potential disease markers [11]. Second, the reduction in complexity achieved by focusing on the glycoprotein subproteome in both tissues and plasma translates into favorable limits of detection, thus increasing the likelihood that the same polypeptide will be detectable in both tissue and serum [8, 12, 13]. Third, aberrant glycosylation is a fundamental characteristic of oncogenesis and tumor progression [14], and this method allows us to identify proteins changed in glycosylation but not necessarily changed in total protein abundance. Finally, specific mass-spectrometry-based methods and affinity reagents can be developed for the specific and sensitive detection of identified tissue proteins in plasma [15], selective isolation of specific proteins or peptides using affinity reagents [16], or the recently developed targeted approach using multiple reaction monitoring (MRM) [1719].

The chemically induced two-stage mouse skin carcinogenesis model has been used for decades to study the genetic, molecular, and biologic basis of tumor development [20]. For example, the concepts of tumor initiation and promotion were derived from this model. In this model, the backs of 8-week-old mice treated with the carcinogen 7,12-dimethylben[a] anthracene (DMBA) followed by multiple treatments with the tumor promoter 12-o-tetradecanoylphorbol-13-acetate (TPA). Benign tumors (papillomas) develop after 8 weeks, and a small percentage of these progresses to malignant invasive carcinomas after a long latency [20]. The ability to quantify both benign and malignant tumor growth permits analysis of genes and environmental factors that affect tumor progression. More recently, the two-stage skin tumor model has been used to improve proteomic technologies for biomarker discovery using serum protein profiling [12]. We have identified several serum proteins for which the abundance is increased in correlation with the chemical induction of skin cancer in mice. However, these proteins are likely not markers for the specific diagnosis of skin cancer. A major advantage of this mouse skin carcinogenesis model is that plasma samples can be taken from mice before and after tumor development. As both benign and malignant tumors and plasma samples can be obtained from the same mice, this facilitates analysis of protein changes in plasma associated with tumor development.

In this paper, we report a two-step strategy for detection of tumor-associated proteins in plasma: the first step was to analyze extracellular proteins from normal skin, papillomas, and carcinomas and identify tumor-associated proteins; the second step was to detect the tumor-associated proteins in plasma using a tissue-targeted approach and isotope labeling [7]. Using our recently developed method of SPEG and mass spectrometry [810], we analyzed matched benign and cancerous tumors from four tumor-bearing mice as well as normal skin tissues from four control mice, and identified 463 unique N-linked glycosites from 318 glycoproteins. More than 40 identified glycoproteins were elevated in carcinomas. Two of the tumor-associated proteins, Tenascin-C and Arylsulfatase B, were further detected and quantified in plasma from the same cancer-bearing mice using isotope labeling and 2D liquid chromatography/matrix-assisted laser desorption/ionization tandem mass spectrometry (LC-MALDI-MS/MS). This result indicates that direct proteomic analysis of tumors and plasma using glycopeptide capture, isotopic labeling, and mass spectrometry can be used to discover new cancer-derived proteins in plasma.

Method and Materials

Materials

Hydrazide resin and sodium periodate were from Bio-Rad (Hercules, CA, USA); PNGase F was from New England Biolabs (Ipswich, MA, USA); sequencing grade trypsin was purchased from Promega (Madison, WI, USA); C18 columns were from Waters (Milford, MA, USA); α-cyano-4-hydroxycinnamic acid (CHCA) was from Agilent (Palo Alto, CA, USA); iTRAQ reagent and mass calibration standards were purchased from Applied Biosystems (Foster City, CA, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Tissues and Plasma from Chemical-Induced Mouse Skin Tumors

Skin tumors were induced in four NIH01a mice using the DMBA/TPA two-step protocol. A single dose of DMBA (Sigma; 25 mg in 200 ml of acetone) was applied to the shaved backs of four 8-week-old mice. Initiated treated skin was promoted with TPA twice a week for 15 weeks. This gave rise to papillomas that were hyperplastic, well-differentiated, benign lesions consisting of keratinocytes together with stroma tissue. Papillomas appeared as early as 8 weeks after the first treatment of DMBA and continued to grow for the next several months. A small percentage of these benign papillomas (~20%) progressed to squamous cell carcinomas. All the mice were killed when carcinomas appeared in all four treated mice. Four littermate mice were left untreated for normal skin tissues. Papillomas and carcinomas, as well as normal skin from untreated mice were snap-frozen in liquid nitrogen. Retro-orbital bleeds were collected from each treated mouse before chemical treatment and after development of chemical-induced carcinomas. The only difference between the normal and cancer tissues is the chemical-induced cancer. Whole blood (0.25 ml) was collected from the retro-orbital sinus into a long (9 in.) sterile glass Pasteur pipet. The whole blood was placed in a K3EDTA-coated 1.5 ml microcentrifuge tube and centrifuged at 4°C for 5 min at 3,000 rpm. Plasma was collected, carefully avoiding cellular contamination. All tumor tissues and plasma were placed in cryovials and frozen in liquid nitrogen.

Peptide Extraction from Skin Tumor Tissues

Frozen tumor tissues (100 mg each) were sliced into 1- to 3-mm3 thickness and incubated in 200 μl of 5 mM phosphate buffer and vortexed for 2–3 min. Then, the samples were sonicated for 5 min in an ice-water bath. Trifluoroethanol (TFE, 200μl) was added to the sample and incubated at 60°C for 2 h followed by sonication for 2 min. Protein disulfide bonds were reduced by 5 mM tributylphosphine with 30-min incubation at 60°C. Iodoacetamide (10 mM) was applied to the mixture and incubated in the dark at room temperature for another 30 min. The samples were diluted fivefold with 50 mM NH4HCO3 (pH 7.8) to reduce the TFE concentration to 10% prior to the addition of trypsin at a ratio of 1:50 (w/w, enzyme/protein). Samples were digested at 37°C overnight with gentle shaking. The precipitate was discarded by centrifuge. Silver staining was used to test the effect of tryptic digestion. Four milligrams of total peptides from each sample was extracted from each tissue. Two milligrams of total peptide was used to extract N-linked glycopeptides, according to the following steps.

Peptide Extraction from Plasma

Plasma (20 μl) was added to 90 μl 8 M urea in 0.4 M NH4HCO3, 0.1% (w/v) sodium dodecyl sulfate (SDS) solution (pH 8.3) and 10 μl 120 mM tris(2-carboxyethyl)phosphine in dH2O freshly prepared and incubated at 60°C for 1 h. Proteins were alkylated by adding 10 μl 160 mM iodoacetamide and incubated at room temperature in the dark with shaking for another 30 min. Samples were diluted by trypsin digestion buffer (100 mM NH4HCO3, pH 8.3) to make the concentration of urea less than 2 M. Forty microliters of trypsin (0.5μg/μl) was added to digest protein at 37°C overnight. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining were employed to check whether trypsin digestion was complete.

Glycopeptide Capture from Tissue or Plasma

N-glycopeptides were isolated from peptides using SPEG [9]. The enriched N-linked glycopeptides were concentrated by C18 columns and dried down and resuspended in 40 μl 0.4% acetic acid prior to MS analysis.

Isotope Labeling of Peptides

The amount of glycopeptide was determined by bicinchoninic acid assay (Bio-Rad) prior to isotope labeling. Glycopeptides (1 μg) from plasma of the retro-orbital bleeds before and after chemical-induced cancer and tumor tissues were dried and resuspended in 20 μl of 50% dimethylformamide, 40% H2O, 10% pyridine. Five microliters 10 mg/ml d013C0, d413C0, and d413C4 succinic anhydride solution was added to glycopeptide samples and reacted at room temperature for 1–2 h, then followed by C18 clean up to remove access succinic anhydride [8].

Mass Spectrometry Analysis

The peptides and proteins were identified using MS/MS analysis using an LTQ ion trap mass spectrometer (Thermo Finnigan, San Jose, CA, USA). Glycopeptides (1 μg) were injected into a peptide cartridge packed with C18 resin and then passed through a 10 cm × 75 μm i.d. microcapillary high-performance liquid chromatography (μLC) column packed with C18 resin. The effluent from the μLC column entered an electrospray ionization source in which peptides were ionized and passed directly into the mass spectrometer. A linear gradient of acetonitrile from 5% to 32% over 100 min at flow rate of ~300 nl/min was applied. During the LC-MS mode, data were acquired between m/z of 400 and 2,000. Each sample was analyzed three times to increase the number of spectra used for spectral count.

Succinic-anhydride-labeled peptide (5 μg) was analyzed by 2D Nano LC (Eksigent, Dublin, CA, USA) and MALDI-tandem time of flight (MALDI-TOF/TOF; Applied Biosystems). Briefly, online integration of 15-cm-long 300 μm strong cation exchange column (SCX) with 15-cm-long 300 μm of C18-reverse phase liquid chromatograph (RPLC) was employed. Four SCX fractions of 0, 5, 50, and 500 mM KCl and 3–45% linear acetonitrile gradient (containing 0.1% TFA and acetonitrile) of RPLC for each fraction were applied before analysis by MALDI-TOF/TOF. Peptides eluted from columns were directly mixed with α-Cyano-4-hydroxycinnamic acid and spotted on a MALDI target plate with 768 spots followed by analysis by MS and MS/MS using ABI4800 MALDI-TOF/TOF.

Data Analyses

Peptide identifications-MS/MS spectra from LTQ were searched with SEQUEST [21] against a mouse protein database (the International Protein Index mouse protein database, version 3.13). The precursor mass tolerance is set as 3.0 Da. Other parameters of database searching are modified as follows: oxidized methionines (add Met with 16 Da), a (PNGase F-catalyzed) conversion of Asn to Asp (add Asn with 1 Da), and Cys modification (add cysteine with 57 Da). The output files were evaluated by INTERACT and PeptideProphet [22, 23]. The criterion of PeptideProphet analysis is the probability score ≥0.9 so that low probability protein identifications can be filtered out.

Identifying tissue-derived peptides in plasma from MALDI-TOF/TOF (ABI 4800) was performed using GPS Explorer software (version 3.6). MS/MS spectra were searched against NCBInr database. GPS searches were carried out at a 0.2-Da precursor mass tolerance, a 0.6-Da fragment mass tolerance, trypsin as digestion enzyme. In addition to the modifications for Met, Asp, and Cys that were used in LTQ MS/MS spectra analyses as described above, N termini of peptides and Lys are modified by succinic anhydride (100 Da for d013C0, 104 Da for d413C0, and 108 Da for d413C4).

Results and Discussion

Strategy of the Method

The objective of this study was to use N-linked glycopeptide isolation, isotopic labeling, and LC-MS to identify skin-cancer-related extracellular proteins and determine if these proteins could be detected in plasma from tumor-bearing mice. This strategy is based on the fact that most extracellular proteins are glycoproteins, and extracellular proteins from cancer are most likely to be detected in plasma due to the fact that they are likely to be secreted by cells or shed from the cell surface to enter the blood stream.

The strategy is schematically illustrated in Fig. 1 and consists of four steps: (1) peptide extraction from tissue or plasma; (2) glycopeptide extraction: peptides that contain N-linked carbohydrates in extracellular proteins were isolated in their de-glycosylated form using a recently described solid-phase capture-and-release method [9, 10]; (3) identification and quantification analysis of glycopeptides isolated from normal skin, papillomas, and carcinomas: isolated peptides were analyzed by LC-MS/MS, and the peptides were identified and quantified using a database search [21] and spectral count; (4) detection of tissue-derived proteins in plasma. Glycopeptides from plasma samples taken from mice before and after development of skin tumors and tumor tissues were labeled with d013C0, d413C0, and d413C4 succinic anhydride, respectively. The peptides containing d413C0 and d413C4 pairs indicated the tumor-derived peptides detected in plasma from tumor-bearing mice, and they were selected for MS/MS analysis for peptide identifications.
Fig. 1

Flow chart for detection of tumor-specific proteins in plasma

Identification of Proteins from a Mouse Model of Skin Cancer

To detect tumor-specific proteins in plasma, we first identified tumor-associated proteins from carcinomas and papillomas. These tumor-associated proteins are likely to be secreted or shed to the blood stream and fall into the detection range of current proteomic methodology.

To identify extracellular proteins from mouse skin tumors, four tissue samples each from normal skin, benign papillomas, and malignant carcinomas were collected to generate pooled normal, benign, and cancer tissues. Proteins were extracted from homogenized frozen tissues and digested to peptides. Glycopeptides were then captured using SPEG from each tissue. The N-linked glycopeptides were analyzed by LC-MS/MS by three repeated analyses for each sample. The MS/MS spectra were used to search protein databases using SEQUEST [21]. There were a total of 4,764 peptide identifications with PeptideProphet of at least 0.9 (with error rate of 0.007) from all the tissues. Ninety percent of these identifications (4,284 identifications) contained a consensus N-linked glycosylation motif (N-X-S/T, X is any amino acid except proline). These identifications were from 463 unique glycosylation sites representing 318 unique glycoproteins (Table 1). This indicated that the procedure was specific to N-linked glycoproteins. Therefore, we limited our subsequent analysis solely to the identified peptide sequences that contained at least one such consensus motif in order to reduce false positive rates. Since tissues are vascularized and some proteins identified from tissues are from contamination by common circulating blood proteins [13, 24], we next examined the glycoproteins identified from tissues to determine glycoproteins identified from tissues that were also identified from the normal mouse plasma [10, 25], and 59 glycoproteins were previously identified from normal mouse plasma and were not included for further study of skin cancer tissues.
Table 1

Identified N-linked glycoproteins and glycosites

IPI

Protein name

P

Identified sequences

IPI00120245

Integrin alpha-V

1

K.AN#TTQPGIVEGGQVLK.C

IPI00120245

Integrin alpha-V

1

R.TAADATGLQPILNQFTPAN#VSR.Q

IPI00127447

Lysosome membrane protein II

1

R.N#QSVGDPNVDLIR.T

IPI00127447

Lysosome membrane protein II

1

T.GEDNYLN#FSK.I

IPI00127447

Lysosome membrane protein II

1

R.TMVFPVMYLN#ESVLIDK.E

IPI00127447

Lysosome membrane protein II

1

R.YKVPAEILAN#TSENAGF.C

IPI00322447

RA175

1

K.VSLTN#VSISDEGR.Y

IPI00322447

RA175

1

R.FQLLN#FSSSELK.V

IPI00118413

Thrombospondin 1

1

L.DNNVVN#GSSPAIR.T

IPI00118413

Thrombospondin 1

1

K.VSCPIMPCSN#ATVPDGECCPR.C

IPI00118413

Thrombospondin 1

1

W.PNENLVCVAN#ATYHCK.K

IPI00123678

Cadherin-22

0.98

R.ETAGWHN#ITVLAMEADN.H

IPI00154057

Protocadherin 1

0.99

N.DNAPFITAPSN#TSHR.L

IPI00126090

Integrin alpha-3

1

I.AMN#YSLPLR.M

IPI00126090

Integrin alpha-3

1

W.LECPLPDTSN#ITN#VTVK.A

IPI00132474

Integrin beta-1

1

R.NPCTSEQN#CTSPFSYK.N

IPI00132474

Integrin beta-1

1

R.KEN#SSEICSNNGECVCGQCVCR.K

IPI00132474

Integrin beta-1

1

K.DTCAQECSHFN#LTK.V

IPI00227969

Integrin alpha-6

1

K.YQTLN#CSVNVR.C

IPI00227969

Integrin alpha-6

0.91

R.VEQKN#NTFFDMNIF.E

IPI00320605

Integrin beta-2

1

K.LN#FTGPGEPDSLR.C

IPI00320605

Integrin beta-2

0.99

Y.LRPGQAAAFN#VTFR.R

IPI00415773

Integrin alpha-M

1

R.TPVLN#CSVAVCK.R

IPI00415773

Integrin alpha-M

1

V.GGPQDFN#MSVTLR.N

IPI00415773

Integrin alpha-M

1

R.LN#YTLVGEPLR.S

IPI00132067

Fibulin-2

1

Y.QLPGCHGN#FSDAEEGDSER.Q

IPI00132067

Fibulin-2

1

K.DLDECALGTHN#CSEAETCHNIQGSFR.C

IPI00132067

Fibulin-2

1

K.SCVAGVMGAKEGETCGAEDN#DTCGVSLYK.A

IPI00223769

CD44 antigen

1

R.TEAADLCQAFN#STLPTMDQMK.L

IPI00110810

Prostate stem cell antigen

1

R.DCLNVQN#CSLDQHSCFTSR.I

IPI00110852

Translocon-associated protein alpha, muscle specific isoform

1

K.DLNGNVFQDAVFN#QTVT.V

IPI00110852

Translocon-associated protein alpha, muscle specific isoform

1

R.YPQDYQFYIQN#FTALPLNTVVPPQR.Q

IPI00112326

Epithelial membrane protein 1

1

K.N#CTGGNCDGSLSYGNEDAIK.A

IPI00113480

Myeloperoxidase

1

R.ALMPFDSLHDDPCLLTN#R.S

IPI00111013

Cathepsin D

1

K.YYHGELSYLN#VTR.K

IPI00111013

Cathepsin D

1

K.N#GTSFDIHYGSGSL.S

IPI00128154

Cathepsin L

1

R.AEFAVAN#DTGFVDIPQQEK.A

IPI00403938

Tenascin-C

1

L.EADTTQTVQN#LTVPGGLR.S

IPI00403938

Tenascin-C

1

R.EPEIGNLN#VSDVTPK.S

IPI00403938

Tenascin-C

1

R.LLQTAEHN#ISGAER.T

IPI00403938

Tenascin-C

1

N.NVEAAQN#LTVPGSLR.A

IPI00403938

Tenascin-C

0.99

N.NVETAHN#FTVPGNLR.A

IPI00403938

Tenascin-C

1

R.ESGLN#MTLPEENQPVVFNHIYNIK.L

IPI00403938

Tenascin-C

1

K.ASTEEVPSLEN#LTVT.E

IPI00403938

Tenascin-C

1

R.LN#YSLPTGQSMEVQLPK.D

IPI00108535

Carcinoembryonic antigen-related cell adhesion molecule 1

1

R.FVPNSNMN#FTGQAYSGR.E

IPI00108535

Carcinoembryonic antigen-related cell adhesion molecule 1

1

K.N#ITVLEPVTQPFLQVTN#TTVK.E

IPI00313428

CEA-related cell adhesion molecule 2

1

R.TLTLLN#VTR.N

IPI00122977

Plasma protease C1 inhibitor

1

R.DTYVN#ASQSLYGSSPR.V

IPI00122977

Plasma protease C1 inhibitor

1

K.VGQLQLSHN#LSFVIVVPVFPK.H

IPI00128689

Collagen alpha 1(V) chain

1

K.VYCN#FTAGGSTCVFPDKK.S

IPI00130249

GPI-anchored metastasis-associated protein homolog

1

A.N#VTVSLPVR.G

IPI00130249

GPI-anchored metastasis-associated protein homolog

1

K.CQGSMPPVVNCYN#ASGR.V

IPI00130486

FK506-binding protein 9

1

R.YHYN#GTLLDGTLFDSSYSR.N

IPI00130486

FK506-binding protein 9

1

R.YHYN#GTFLDGTLFDSSHNR.M

IPI00132600

Niemann-Pick C1 protein

1

R.LYN#VTHQFCN#ASVMDPTCVR.C

IPI00132600

Niemann-Pick C1 protein

1

R.LIASN#ITETMR.S

IPI00131881

ADAM 10

1

R.IN#TTSDEKDPTNPFR.F

IPI00130342

Lymphocyte antigen 6 complex locus G6C protein

1

K.LGLNYN#TTCCDK.D

IPI00130342

Lymphocyte antigen 6 complex locus G6C protein

1

R.EVFN#ETNHK.L

IPI00133082

CD177 antigen

1

K.VQGCMAQPDCNLLN#GTQAI.G

IPI00134549

Lysosome-associated membrane glycoprotein 2

1

A.LIVN#LTDSK.G

IPI00134549

Lysosome-associated membrane glycoprotein 2

1

K.VPFIFNINPATTN#FTGSCQPQSAQLR.L

IPI00134549

Lysosome-associated membrane glycoprotein 2

1

K.EVNVYMYLAN#GSAFN#ISNK.N

IPI00121430

Collagen alpha 1(XII) chain

1

K.EAGN#ITTDGYEILGK.L

IPI00122272

Extracellular matrix protein 1

1

K.QIPGLIQN#MTVR.C

IPI00122272

Extracellular matrix protein 1

1

R.NVALVAGDTGN#ATGLGEQGPTR.G

IPI00122493

FK506-binding protein 10

1

R.YHYN#CSLLDGTR.L

IPI00122493

FK506-binding protein 10

1

R.YHYN#GSLMDGTLFDSSYSR.N

IPI00123342

Hypoxia up-regulated 1

1

R.VFGSQN#LTTVK.L

IPI00123342

Hypoxia up-regulated 1

1

R.LSALDNLLN#HSSIFLK.G

IPI00123342

Hypoxia up-regulated 1

1

K.EN#GTDAVQEEEESPAEGSK.D

IPI00123831

SDR1 protein

1

K.ENGVFEEISN#SSGR.F

IPI00123831

SDR1 protein

1

R.FFITNKEN#YTEL.S

IPI00123831

SDR1 protein

1

R.ESLLPVTLQCN#LTSSSH.T

IPI00224728

Cd63 antigen

1

K.DRVPDSCCIN#ITVGCGNDFK.E

IPI00462199

Basigin

1

K.TSDTGEEEAITN#STEANGK.Y

IPI00462199

Basigin

1

K.TQLTCSLN#SSGVDIVGHR.W

IPI00462199

Basigin

1

K.SQLTISNLDVNVDPGTYVCN#ATNAQGTTR.E

IPI00308609

VESICULAR INTEGRAL-MEMBRANE PROTEIN VIP36

1

R.VFPYISVMVNN#GSLSYDHSK.D

IPI00308990

Monocyte differentiation antigen CD14

1

R.N#PSPDELPQVGN#LSLK.G

IPI00308785

Prostaglandin G/H synthase 2

1

R.TGFYGEN#CTTPEFLTR.I

IPI00308971

Cation-independent mannose-6-phosphate receptor

1

K.ISTN#ITLVCKPGDLESAPVLR.A

IPI00308971

Cation-independent mannose-6-phosphate receptor

1

R.SLLEFN#TTMGCQPSDSQHR.I

IPI00124836

Beta-sarcoglycan

1

R.ITSN#ATSDLNIK.V

IPI00124836

Beta-sarcoglycan

0.99

I.ILN#GTVMVSPTR.L

IPI00122737

222 kDa protein

1

R.QAEEAEEQANTN#LSK.F

IPI00122737

222 kDa protein

0.98

R.VQLLHSQN#TSLINQKK.K

IPI00119063

AM2 receptor

1

K.LTSCATN#ASMCGDEAR.C

IPI00119063

AM2 receptor

1

K.LNLDGSN#YTLLK.Q

IPI00119063

AM2 receptor

1

A.VAN#DTNSCELSPCR.I

IPI00119063

AM2 receptor

1

R.MGCQHHCVPTPSGPTCYCN#SSFQLE.A

IPI00119063

AM2 receptor

0.99

R.GVTHLN#ISGLK.M

IPI00119063

AM2 receptor

1

R.FN#STEYQVVTR.V

IPI00124265

Latent transforming growth factor beta binding protein 4

1

R.N#ATSVDSGAPGGAAPGGPGFR.A

IPI00124265

Latent transforming growth factor beta binding protein 4

1

R.CTPACDPGYQPTPGGGCQDVDECRN#R.S

IPI00129304

Collectin sub-family member 12

1

R.HTDDLTSLN#NTLVNIR.L

IPI00129304

Collectin sub-family member 13

1

K.ETLQN#NSFLITTVN#K.T

IPI00153959

Stabilin-1

1

H.ADLISN#MSQDELAR.I

IPI00153959

Stabilin-1

1

K.GFVDN#MTLSGPDLELH.A

IPI00316575

Cathepsin K

1

Y.VGQDESCMYN#ATAK.A

IPI00126769

Cathepsin F

0.94

K.VYIN#DSVELSR.N

IPI00121190

Epidermal growth factor receptor

1

R.DIVQNVFMSN#MSMDLQSHPSSCPK.C

IPI00320420

Clusterin

1

R.QELN#DSLQVAER.L

IPI00320420

Clusterin

0.99

K.MLN#TSSLLEQLNDQFNWVSQLAN#LTQGEDK.Y

IPI00406459

Arylsulfatase B

1

H.EACAPIESLN#GTR.C

IPI00406459

Arylsulfatase B

1

R.IYAGMVSLMDEAVGN#VTK.A

IPI00409393

Latent transforming growth factor beta binding protein, isoform 1L

1

R.YGQEQGTAPFQVSN#HTGR.I

IPI00409393

Latent transforming growth factor beta binding protein, isoform 1L

1

Y.NLNDASLCDNVLAPN#VTK.Q

IPI00409393

Latent transforming growth factor beta binding protein, isoform 1L

0.91

K.VCTN#GSCTNLEGSYM.C

IPI00108535

Carcinoembryonic antigen-related cell adhesion molecule 1

1

R.EIIYSN#GSLLFQMITMK.D

IPI00117424

Intercellular adhesion molecule 2

1

K.IN#CSTNCAAPDMGGLETPTNK.I

IPI00122971

N-CAM 180 of Neural cell adhesion molecule 1, 180 kDa isoform

1

R.DGQLLPSSN#YSNIK.I

IPI00406901

Platelet/endothelial cell adhesion molecule

1

K.EETVLSQYQN#FSK.I

IPI00115976

Integrin alpha-5

1

K.VTGLSN#CTSN#YTPN.S

IPI00313479

Integrin beta 4 Isoform 2

1

K.TCN#CSTGSLSDTQPCLR.E

IPI00466371

Integrin alpha 1

1

K.DSCESNQN#ITCR.V

IPI00230432

Fibulin-1

0.98

H.SYN#SSLETIFIK.R

IPI00119756

OX-2 membrane glycoprotein

1

K.GTGTGIEN#STESHFHSN#GTTSVTSILR.V

IPI00222589

PTK7 protein tyrosine kinase 7

0.98

R.MHIFQN#GSLVIH.D

IPI00314779

TGF-beta receptor type III

1

R.AGVVVFN#CSLR.Q

IPI00112787

Cell surface glycoprotein OX2 receptor

1

W.SPDGDCVTTSESHSN#GTVTVR.S

IPI00113528

Transmembrane 9 superfamily protein member 3

1

R.IVDVN#LTSEGK.V

IPI00114304

Thrombospondin-3

1

R.LGFLGN#QSQGCVPAR.T

IPI00119809

Mama protein

1

R.ALGYEN#ATQALGR.A

IPI00119809

Mama protein

1

K.GLN#LTEDTYKPR.L

IPI00469218

Lysosomal membrane glycoprotein 1

1

R.LN#MTLPDALVPTFSISN#HSLK.A

IPI00469218

Lysosomal membrane glycoprotein 2

1

K.N#VTVVLR.D

IPI00120025

Similar to KALLIKREIN 9

1

R.LTPAVQPLN#LTESRPPVGTQ.C

IPI00338790

Glandular kallikrein KLK13

1

K.ILN#GTN#GTSGFLPGGYTCLPH.S

IPI00116993

Tuberin

0.94

A.PKQGLN#NSPPVK.E

IPI00111550

Mucin and cadherin-like protein

1

R.VTN#SSEFMMNK.D

IPI00108041

Stromal interaction molecule 1

1

R.LAVTN#TTMTGTVLK.M

IPI00108328

Methylated-DNA- protein-cysteine methyltransferase containing protein

0.93

M.ETTSLLLCIGN#NSSGIRSRHR.S

IPI00108811

Glucosylceramidase

1

R.DLGPALAN#SSHDVK.L

IPI00109281

Enabled protein homolog

1

W.ERTNTMN#GSK.S

IPI00109612

Laminin, beta 2

1

L.ASGN#VSGGVCDGCQHNTAGR.H

IPI00109727

Thy-1 membrane glycoprotein

1

K.VLTLAN#FTTK.D

IPI00109908

Ig gamma-2A chain C region, membrane-bound form

1

R.EDYN#STLR.V

IPI00111014

Elongation of very long chain fatty acids protein 4

1

T.AFN#DTVEFYR.W

IPI00111115

Similar to METASTASIS-ASSOCIATED GPI- ANCHORED PROTEIN

1

R.MNIGN#FSVPVYIR.T

IPI00111960

Lysosomal alpha-glucosidase

1

R.GVFITN#ETGQPLIGK.V

IPI00112176

Copper homeostasis protein cutC homolog

0.94

R.N#SSVAMGASLAHSEYSLK.V

IPI00113057

Plasma kallikrein

1

K.LQTPLN#YTEFQKPICLPSK.A

IPI00113797

Napsin A

0.96

W.FN#LTGQDYVIK.I

IPI00113824

Basement membrane-specific heparan sulfate proteoglycan core protein

0.98

K.LTVPSSQN#SSFR.L

IPI00113824

Basement membrane-specific heparan sulfate proteoglycan core protein

1

R.SLTQGSLIVGNLAPVN#GTSQGK.F

IPI00113824

Basement membrane-specific heparan sulfate proteoglycan core protein

1

R.VAQQDSGQYICN#ATNSAGH.T

IPI00113853

Desmocollin-3

0.99

K.AN#FTILK.G

IPI00113854

Eosinophil peroxidase

0.99

F.DNLHEDPCLLTN#R.S

IPI00114065

Complement factor B

1

K.IVLDPSGSMNIYLVLDGSDSIGSSN#FTGAK.R

IPI00114065

Complement factor B

0.94

R.SPFYN#LSDQI.S

IPI00114206

Prothrombin

1

R.WVLTAAHCILYPPWDKN#FTENDLLVR.I

IPI00114206

Prothrombin

1

R.ITDNMFCAGFKVN#DTK.R

IPI00400016

Laminin gamma-1 chain

1

K.LLNN#LTSIK.I

IPI00400016

Laminin gamma-1 chain

1

R.TLAGEN#QTALEIEELNR.K

IPI00400016

Laminin gamma-1 chain

1

L.SYGQN#LSFSFR.V

IPI00400016

Laminin gamma-1 chain

1

R.KYEQAKN#ISQDLEKQ.A

IPI00317340

Lactotransferrin

1

I.PMGLLAN#QTR.S

IPI00317340

Lactotransferrin

1

K.N#SSNFHLNQLQGLR.S

IPI00113539

Fibronectin

1

R.DQCIVDDITYNVN#DTFHK.R

IPI00113539

Fibronectin

1

K.LDAPTNLQFVN#ETDR.T

IPI00113539

Fibronectin

1

R.HEEGHMLN#CTCFGQGR.G

IPI00119818

Inter alpha-trypsin inhibitor, heavy chain 4

1

K.AFITN#FSMIIDGVTYPGVVK.E

IPI00119818

Inter alpha-trypsin inhibitor, heavy chain 5

1

R.GLMLLLN#DTQHFSNNVK.G

IPI00114256

Synaptophysin-like protein

1

K.N#QTVTATFGYPFR.L

IPI00114319

Extracellular superoxide dismutase [Cu-Zn]

1

R.LEAYFSLEGFPAEQN#ASNR.A

IPI00114641

CD98 heavy chain

1

K.LMNAPLYLAEWQN#ITK.N

IPI00114810

Suppressor of tumorigenicity 14

0.99

R.VIN#QTTCEDLMPQQITPR.M

IPI00114958

HMW of Kininogen-1

1

K.HSIEHFNN#NTDHSHLFTLR.K

IPI00114958

HMW of Kininogen-1

1

T.YTIVQTN#CSK.E

IPI00114958

HMW of Kininogen-1

1

K.IAN#FSQSCTLYSGDDLVEALPKPCPGCPR.D

IPI00115089

Ectonucleoside triphosphate diphosphohydrolase 2

1

R.LLN#LTSPEATAK.V

IPI00115516

EMILIN-1

1

R.FN#STLGPSEEQEK.N

IPI00115530

Beta-hexosaminidase beta chain

1

K.TQVFGPVDPTVN#TTYA.F

IPI00115762

Neural cell adhesion molecule L1

1

K.EQLFFN#LSDPELR.T

IPI00115817

PREDICTED: similar to ribosomal protein L21

0.95

K.TGRVYN#VTQHAMGIIVNK.Q

IPI00115854

TROP2 protein

1

R.AFN#HSDLDSELR.R

IPI00116105

Corticosteroid-binding globulin

1

K.DLFTN#QSDFADTTK.D

IPI00116105

Corticosteroid-binding globulin

1

R.EEDFYVN#ETSTVK.V

IPI00116105

Corticosteroid-binding globulin

1

K.VPMMVQSGN#ISYFR.D

IPI00116105

Corticosteroid-binding globulin

1

R.GSTQYLENLGFN#MSK.M

IPI00116599

p130Cas-associated protein

0.92

R.RQVDEGMWPPPNNLLN#QSPK.K

IPI00116913

Laminin alpha-5 chain

1

R.QLLAN#SSALEETILGHQGR.L

IPI00116913

Laminin alpha-5 chain

1

H.N#FSGCISNVFVQR.L

IPI00116945

Complement factor D

1

K.LSQN#ASLGPHVRPLPLQYEDK.E

IPI00117093

Laminin beta-3 chain

1

R.QTACTPGDCPGELCPQDN#GTACGSHCR.G

IPI00117140

Fc receptor, IgG, low affinity IIb

1

R.YHHYSSN#FSIPK.A

IPI00117735

Myelin P0 protein

1

K.DGSIVIHNLDYSDN#GTFTCDVK.N

IPI00117831

Ceruloplasmin

1

K.EYEGAVYPDN#TTDFQR.A

IPI00117857

Alpha-1-antitrypsin 1-6

1

K.GDTHTQILEGLQFN#LTQTSEADIHK.S

IPI00117932

Paired amphipathic helix protein Sin3a

0.96

P.DAN#SSVLLSKTTAEK.V

IPI00117957

Asporin

1

R.ITDIEN#GTFANIPR.V

IPI00118011

mannosidase, beta A, lysosomal

0.97

V.AEILFNN#VTIGK.T

IPI00118130

Alpha-1-acid glycoprotein 1

1

R.ESQTIGDQCVYN#STHLGFQR.E

IPI00118130

Alpha-1-acid glycoprotein 1

1

R.QAIQTMQSEFFYLTTNLIN#DTIELR.E

IPI00118130

Alpha-1-acid glycoprotein 1

1

R.EN#GTFSKYEGGVETFAHLIVLR.K

IPI00118191

Receptor-type tyrosine-protein phosphatase N2

0.98

K.VSANIQN#MTTADVIK.A

IPI00118385

Glutamate [NMDA] receptor subunit zeta 1

1

K.VICTGPN#DTSPGSPR.H

IPI00118437

Complement component C8 gamma chain homolog

1

R.EAN#LTEDQILFFPK.Y

IPI00119004

Hypothetical Lipolytic enzyme, G-D-S-L containing protein

0.91

R.KGPGMENPVAVTIFFGAN#DSSLK.D

IPI00119299

Leukemia inhibitory factor receptor

1

K.VVLAGSN#MTICCMSPTK.V

IPI00119299

Leukemia inhibitory factor receptor

1

R.IEGLTN#ETYR.L

IPI00119299

Leukemia inhibitory factor receptor

1

R.LGVQMHPGQEIHN#FTLTGR.N

IPI00119522

Carboxypeptidase N, polypeptide 2 homolog

1

R.LQDLEITGSPVSN#LSAHIFSN#LSSLEK.L

IPI00119627

Insulin receptor substrate 1

0.93

K.LLPCTGDYMN#MSPVGDSN#TS.S

IPI00120187

Fibromodulin

0.97

R.VPNNALEGLEN#LT.A

IPI00120751

Proton myo-inositol transporter homolog

1

K.IN#GSAVIDSSCVPVNK.A

IPI00120769

Solute carrier family 29 (nucleoside transporters), member 1

1

R.LDVSQN#VSSDTDQSCESTK.A

IPI00120848

Mimecan

0.95

I.SSLTDDTFCKAN#DTR.Y

IPI00121038

Versican core protein

1

R.FEN#QTCFPLPDSR.F

IPI00121120

Procollagen, type V, alpha 2

1

K.EASQN#LTYICR.N

IPI00121312

MFIRE1

1

K.IDLTDFEKN#SSFA.Q

IPI00121362

F11r protein

1

R.AFMN#SSFTIDPK.S

IPI00121418

Retinoblastoma-associated protein

0.97

K.QLEN#DTRIIEVLCKEHECNIDEVKN.V

IPI00121550

Sodium/potassium-transporting ATPase beta-1 chain

1

K.LDWLGN#CSGLNDDSYGYR.E

IPI00121634

High-affinity cationic amino acid transporter-1

0.94

K.FLAKINN#RTKTPVIATVTSGAIAAVM.A

IPI00122293

Prolargin

1

R.VPVIPPRIHYLYLQNNFITELPLESFQN#ATGLR.W

IPI00122302

Neutrophil elastase homolog

0.91

R.LGTNRPSPSVLQELN#VT.V

IPI00122368

P2X4c receptor subunit

1

K.TSICDSDAN#CTLGSSDTHSSGIGTGR.C

IPI00122438

Fibrillin-1

1

K.AWGTPCELCPSVN#TSEYK.I

IPI00122438

Fibrillin-1

1

V.DTDECSVGNPCGN#GTCK.N

IPI00122438

Fibrillin-1

1

V.N#VTDYCQLVR.Y

IPI00122438

Fibrillin-1

1

R.NYYADN#QTCDGELLFN#MTK.K

IPI00122438

Fibrillin-1

1

R.N#CTDIDECR.I

IPI00123194

Biglycan

1

R.MIEN#GSLSFLPTLR.E

IPI00123196

Decorin

1

K.LGLSFNSITVMEN#GSLANVPHLR.E

IPI00123196

Decorin

1

K.YIQVVYLHNNN#ISAVGQNDFCR.A

IPI00123223

Murinoglobulin-1

1

R.NYEVQLFHVN#ATVTEEGTGLEFSR.S

IPI00123223

Murinoglobulin-1

1

R.N#ASFVYTK.A

IPI00123824

Amiloride-sensitive sodium channel beta-subunit

1

K.GEPYSPCTMN#GSDVAIK.N

IPI00123957

Cd97 protein

1

R.DFNPATVN#YTIQK.L

IPI00123996

Neuropilin-1

1

K.RGPECSQN#YTAPTGVIK.S

IPI00124283

Macrophage scavenger receptor types I and II

1

R.VLNN#ITNDLR.L

IPI00124640

Osteoclast-like cell cDNA, clone:I420031M06 product:granulin

1

K.SDTPCDDFTRCPTN#NTCCK.L

IPI00124830

Leukocyte surface antigen CD47

1

I.EFTSCN#ETVVIPCIVR.N

IPI00125058

Laminin alpha-3 chain

1

K.IESINQQLLPLGN#ISDNVDR.I

IPI00125058

Laminin alpha-3 chain

0.99

K.TTFNLN#TTEVEPCRR.R

IPI00125266

Acid ceramidase

1

R.SVLEN#TTSYEEAK.N

IPI00125293

Eosinophil cationic protein 1

0.97

R.VHITVCN#ITSR.A

IPI00125310

Complement C1q subcomponent, A chain

1

K.VLTNQESPYQN#HTGR.F

IPI00125325

Peroxisomal 2,4-dienoyl-CoA reductase

0.96

F.RDHGGVIVN#ITATLSMR.G

IPI00125514

Ectonucleoside triphosphate diphosphohydrolase 5

1

R.GYLTSFEMFN#STFK.L

IPI00125877

Hypothetical protein

1

N.YQN#NTEVIQGIR.T

IPI00125877

Hypothetical protein

1

R.GLTFLKN#VSSTCAASPSTDILTFTIPPSFADIFLSK.S

IPI00126050

Plasma glutamate carboxypeptidase

1

K.EVMNLLQPLN#VTK.V

IPI00126186

Macrophage mannose receptor 1

1

R.TSYCN#ESFYFLCK.K

IPI00126194

Alpha-2-macroglobulin

1

K.N#ITSVVSPLGYLSIFTTDEHGLAN#ISIDTSN#FTAPFLR.V

IPI00126194

Alpha-2-macroglobulin

1

R.IN#VSYTGERPSSNMVIVDVK.M

IPI00126194

Alpha-2-macroglobulin

1

Y.LN#ETQQLTEAIK.S

IPI00126194

Alpha-2-macroglobulin

1

K.VN#LSFPSAQSLPASDTHLK.V

IPI00126316

Mast cell carboxypeptidase A

1

R.NQN#STCIGTDLNR.N

IPI00126834

Vascular cell adhesion protein 1

1

K.ETTIWVSPSPILEEGSPVN#LTCSSDGIPAPK.I

IPI00127280

Myeloid bactenecin

1

K.DCDFLEDGEERN#CTGK.F

IPI00127352

AMBP protein

1

K.EDSCQLN#YSEGPCLGMQER.Y

IPI00127560

Transthyretin

1

K.TLGISPFHEFADVVFTAN#DSGHR.H

IPI00127672

PREDICTED: hypothetical protein LOC66967

1

K.LLPAFN#TTSGLPYPR.I

IPI00127856

Alpha-1-acid glycoprotein 2

1

R.EYHTIDDHCVYN#STHLGIQR.E

IPI00127856

Alpha-1-acid glycoprotein 2

1

D.PITN#ETLSWLSDK.W

IPI00127933

Androgen binding protein alpha

1

R.KVDLFLN#GTTEEY.V

IPI00128249

Alpha-2-HS-glycoprotein

1

R.RPFGVVYEMEVDTLETTCHALDPTPLAN#CSVR.Q

IPI00128249

Alpha-2-HS-glycoprotein

1

R.CPLLTPFN#DTNVVHTVNTALAAFNTQNN#GTYFK.L

IPI00128484

Hemopexin

1

R.VAEVEN#GTKPD.S

IPI00128484

Hemopexin

1

R.SWSTVGN#CTAALR.W

IPI00128484

Hemopexin

1

K.SLGPNTCSSN#GSSLYFIHGPNLYCYSSIDK.L

IPI00128484

Hemopexin

1

M.DHN#GTMLFFK.G

IPI00128905

Golgi phosphoprotein 2

1

K.AVLVNN#ITTGEK.L

IPI00128989

Vacuolar ATP synthase subunit S1

1

A.IHPPVSYN#DTAPR.I

IPI00129158

Tyrosine-protein phosphatase non-receptor type substrate 1

1

R.GIAN#LSNFIR.V

IPI00129243

Gamma-glutamyl hydrolase

1

K.LPLN#FTEGAR.K

IPI00129243

Gamma-glutamyl hydrolase

0.99

L.ALEN#LTANFHK.W

IPI00129250

Leucine-rich alpha-2-glycoprotein

1

L.SVEFSN#LTQLPAAALQGCPGLR.E

IPI00129250

Leucine-rich alpha-2-glycoprotein

1

K.MFSQN#DTR.C

IPI00129359

zinc finger protein 68

0.97

K.ELAGIGNTCN#VSTNH.I

IPI00129965

PREDICTED: similar to alpha-1-B glycoprotein

1

K.LLFVGPQHAGN#YSCR.Y

IPI00129966

PREDICTED: similar to alpha-1-B glycoprotein

0.99

R.VYQPGN#YSCSYQTHGECTSSTPSR.I

IPI00129968

Embigin

1

K.DDEPLETTGDFN#TTK.M

IPI00130010

Complement factor H

1

K.DNSCVDPPHVPN#ATIVTR.T

IPI00130010

Complement factor H

1

K.LTEFTHN#STMDYK.C

IPI00130010

Complement factor H

1

R.TKCIN#GTINYPTCV.-

IPI00130015

Dipeptidyl-peptidase I

1

R.ILTN#NSQTPILSPQEVVSCSPYAQGCDGGFPYLIAGK.Y

IPI00130483

KH domain RNA binding protein QKI-5A

0.96

R.KDMYN#DTLN#GSTEK.R

IPI00130627

Legumain

0.97

Y.DDIANSEEN#PTPGVVINRPN#GTDVYK.G

IPI00130630

Glutamate carboxypeptidase II

1

K.VPYNVGPGFAGN#FSTQK.V

IPI00130654

Afamin

1

P.TKPQDVDHFN#ATQK.F

IPI00130654

Afamin

1

L.ADLVLGELCGVNTN#R.T

IPI00130661

Tripeptidyl-peptidase I

0.97

K.DVGSGTTN#NSQACAQFLEQYFHNSDLTEFMR.L

IPI00130661

Tripeptidyl-peptidase I

1

K.SSSHLPPSSYFN#ASGR.A

IPI00131114

Type VI collagen alpha 3 subunit

1

R.GPPGVN#GTQGFQGCPGQR.G

IPI00131114

Type VI collagen alpha 3 subunit

1

R.ALN#GSALYTGSSLDFVR.N

IPI00131114

Type VI collagen alpha 3 subunit

1

R.QLINALQIN#NTAVGHALVLPAR.R

IPI00131137

9 kDa protein

0.96

K.GKAN#ASEDANNPAENGDAK.T

IPI00131209

Keratin intermediate filament 16a

1

R.KTEELNKEVASNSDLIQSN#R.S

IPI00131366

Keratin, type II cytoskeletal 6B

1

R.VPGLN#RSGFSSVSVCR.S

IPI00131526

CD209 antigen-like protein B

1

R.IPIFQGQN#ESIQEK.I

IPI00131830

Serine protease inhibitor A3K

1

K.NLINDYVSN#QTQGMIK.E

IPI00131830

Serine protease inhibitor A3K

1

K.YTGN#ASALLILPDQGR.M

IPI00131951

Serpin A12

0.91

L.SLGAQN#STLEEIR.E

IPI00133035

NAD(P)(+)-arginine ADP-ribosyltransferase

1

R.LGN#FTLAYSAKPETADNQR.V

IPI00133035

NAD(P)(+)-arginine ADP-ribosyltransferase

1

K.GTSNDLVLQSIN#STCSYYECAFLGGLK.T

IPI00133172

Serpin B11

1

K.N#SSECSQVGVMHPDFR.A

IPI00133257

Hematopoietic progenitor cell antigen CD34

1

M.VLAN#STELPSK.L

IPI00133751

Microfibril-associated glycoprotein 4

1

R.VDLEDFEN#NTAYAK.Y

IPI00133751

Microfibril-associated glycoprotein 4

1

R.FN#GSVSFFR.G

IPI00134191

Solute carrier family 2, facilitated glucose transporter member 3

1

K.DFLN#YTLEER.L

IPI00134483

Lectin lambda

1

R.PGACTN#ITMGVVCK.L

IPI00134483

Lectin lambda

1

R.VTPVCN#ASLPAQR.W

IPI00134547

Zinc finger autosomal protein

0.98

V.ELLDPN#NSICVPREK.M

IPI00134652

Type VII collagen

1

K.LQILN#ASSDVLR.V

IPI00134808

C4b-binding protein

1

R.LACLN#GTVLR.G

IPI00134808

C4b-binding protein

1

R.LVGSPFIGCTVVN#K.T

IPI00136642

Antithrombin-III

1

K.LGACN#DTLK.Q

IPI00136902

Piccolo protein

0.97

Y.RRQISAVQPSIIN#LSAASSLGTPVTMDSK.T

IPI00136925

Immunoglobulin J chain

1

R.EN#ISDPTSPLR.R

IPI00137177

Lysosomal protective protein

1

R.LDPPCTN#TTAPSNYLNNPYVR.K

IPI00137987

Zinc-alpha-2-glycoprotein

1

K.DTTGSHTFQGMFGCEITNN#R.S

IPI00138342

Liver carboxylesterase N

1

R.FHSELN#ISESMIPAVIEK.Y

IPI00139788

Serotransferrin

1

K.N#STLCDLCIGPLK.C

IPI00153187

Sulfatase modifying factor 1

1

K.FVN#STGYLTEAEK.F

IPI00153202

Angiotensin-converting enzyme 2

0.99

Y.FFVTSPQN#VSDVIPR.S

IPI00153258

Protein Z-dependent protease inhibitor

1

R.ASQQLSN#ETSSFGFNLLR.K

IPI00153548

Hypothetical protein

0.9

C.QFGVGTFANVFLFVYN#FSPISTGSK.Q

IPI00169815

Procollagen, type VI, alpha 2

1

R.GTFTDCALAN#MTQQIR.Q

IPI00169815

Procollagen, type VI, alpha 2

1

I.GYTN#FTLEK.N

IPI00169815

Procollagen, type VI, alpha 2

1

R.MALLQYGSQNQQQVAFPLTYN#VTTIHEALER.A

IPI00169815

Procollagen, type VI, alpha 2

1

R.N#MTLFSDLVAEK.F

IPI00169858

Hypothetical protein LOC435366

0.97

R.HERN#QSAEKPSEYTQHGKAFALHAHSHAQ.R

IPI00169896

Choline transporter-like protein 2

1

K.TCNPETFPLRN#ESLQCPTAR.C

IPI00221418

Hypothetical Phospholipase D/Transphosphatidylase

1

K.VFIVPVGN#HSNIPFSR.V

IPI00221426

Glucosamine (N-acetyl)-6-sulfatase

1

K.YYN#YTLSINGK.A

IPI00221456

Synaptic vesicle glycoprotein 2 b

0.96

K.KVLSMSLAIN#ASFASLSSFVQGY.G

IPI00221833

Hypothetical Zinc finger, C2H2 type containing protein

0.96

D.WMPNN#HSVILIDDFESPQK.L

IPI00223446

Laminin alpha-4 chain

1

R.HVTDMN#STIHLLR.T

IPI00223987

Insulin-regulated membrane aminopeptidase IRAP homolog

1

R.MAFDLIDYLKN#ETHTAPI.T

IPI00224456

Sarcalumenin

1

A.PLIN#VTEPPR.V

IPI00224456

Sarcalumenin

1

K.TN#VSKFDLPNR.E

IPI00224584

Calsequestrin 2

1

K.IDLFKPQIGVVN#VTDADSI.W

IPI00224654

Hypothetical protein

1

R.AYIQDFQEFSKN#ISIMLGR.C

IPI00225355

Target of Nesh-SH3 variant 1

0.99

K.VHIN#TTSDSILLK.F

IPI00226310

Hypothetical von Willebrand factor type A domain containing protein

1

R.DLSVFAPN#MTEIIK.D

IPI00226310

Hypothetical von Willebrand factor type A domain containing protein

1

K.LGN#FSELATHN#QTFLK.K

IPI00226310

Hypothetical von Willebrand factor type A domain containing protein

0.99

L.LDMAIN#GSQEDLDHLK.A

IPI00226790

GPI transamidase component PIG-T

0.92

L.GLAN#DTDDYFLR.Y

IPI00226932

Quinoprotein alcohol dehydrogenase structure containing protein

1

R.FINYN#QTVSR.M

IPI00227834

Inter-alpha trypsin inhibitor, heavy chain 2

1

K.GAFISN#FTMTVNGMTFTSSIK.E

IPI00227857

Hepatocyte growth factor activator

1

R.FCNIVPTEHCFLGN#GTEYR.G

IPI00229117

Tenascin-N

1

Y.ILTYQFPN#GTVK.E

IPI00230289

Excitatory amino acid transporter 2

1

K.VLVAPPSEEAN#TTK.A

IPI00266902

PREDICTED: similar to type V P-type ATPase isoform 3

0.99

K.VCDPNSDVCN#TTR.S

IPI00271166

Huntington disease gene homolog

0.94

R.GYSLLPSITDVTMENN#LSR.V

IPI00271262

Murinoglobulin-2

1

K.ELIFYYLVMAQGSIIQTGN#HTHQVEPGEAPVK.G

IPI00272381

Proline 4-hydroxylase, alpha 1

1

K.DMSDGFISN#LTIQR.Q

IPI00279010

Lu protein

1

F.VFLN#SSSTVVN#CSAR.G

IPI00279051

RIKEN cDNA A930025J12

1

R.LFQN#CSELYK.A

IPI00279079

Fibrinogen beta chain

1

K.GTAGNALMDGASQLVGEN#R.T

IPI00281188

140 kDa protein

0.99

K.VLEPPHIN#GSEGPGEV.S

IPI00281344

Hypothetical Glycosyl transferase, family 8 containing protein

0.93

R.TGVNSGVMLMN#MTR.M

IPI00308213

Ig gamma-1 chain C region, membrane-bound form

1

R.EEQFN#STFR.S

IPI00308658

Olfactomedin-like protein 3

1

K.IYVLDGTQN#DTAFVFPR.L

IPI00309068

E130014G12 product:Kaiso protein

1

K.EDLPSN#NT.A

IPI00309214

Serum amyloid P-component

1

K.LIPHLEKPLQN#FTLCFR.T

IPI00309230

Beta-glucuronidase

0.98

R.ITIAIN#NTLTPH.T

IPI00309999

Laminin alpha-2 chain

1

R.LEQMTMNIN#LTGPLPAPYK.I

IPI00309999

Laminin alpha-2 chain

1

K.LN#ETLGNQDK.T

IPI00309999

Laminin alpha-2 chain

1

R.ICNQN#SSNPYQR.H

IPI00309999

Laminin alpha-2 chain

1

K.VFQAESHAAQLN#DSSAVLDGILDEAK.N

IPI00309999

Laminin alpha-2 chain

1

K.VCN#CSTVGSLASQCNVNTGQCSCHPK.F

IPI00309999

Laminin alpha-2 chain

1

K.ILYGLEN#TTQELK.H

IPI00309999

Laminin alpha-2 chain

1

K.YIGGGVCIN#CTHNTA.G

IPI00309999

Laminin alpha-2 chain

1

Y.VGGLPIN#YTTR.R

IPI00309999

Laminin alpha-2 chain

1

L.NLASNALITTN#ATCGEK.G

IPI00310049

Carboxypeptidase B2

1

K.EVHFFVN#ASDVDSVK.A

IPI00311808

Transmembrane glycoprotein NMB

1

R.DLPIVFDVLIHDPSHFLN#DSAISYK.W

IPI00313900

Lumican

1

K.LHINYNN#LTESVGPLPK.S

IPI00313900

Lumican

1

R.LSHNELADSGVPGNSFN#ISSLLELDLSYNK.L

IPI00313900

Lumican

1

K.AFEN#VTDLQWLILDHNLLENSK.I

IPI00313900

Lumican

1

K.LGSFDGLVN#LTFIYLQHNQLK.E

IPI00316329

keratin complex 2, basic, gene 1

1

R.MSGECTPN#VSVSVSTSHTSMSGSSSR.G

IPI00318012

T-cell immunomodulatory protein

1

V.PCNN#ASCEEVHR.M

IPI00318595

Adipocyte-derived leucine aminopeptidase

1

L.N#SSHPVSTPVENPAQIR.E

IPI00319814

Suprabasal-specific protein suprabasin

1

K.EANQLLN#GSHQGQGGYGGQHGGAATT.T

IPI00320204

RIKEN cDNA 2210023G05

1

R.DGTSQPAICPQN#VTMNMEGLK.E

IPI00320675

Complement factor I

1

R.WGEVDLIGN#CSQFYPDR.Y

IPI00320675

Complement factor I

1

N.FN#VSLIYGR.T

IPI00321190

Sulfated glycoprotein 1

1

K.TN#SSFIQGFVDHVKEDCDR.L

IPI00321190

Sulfated glycoprotein 1

1

K.DN#ATQEEILHYLEK.T

IPI00322304

Histidine-rich glycoprotein HRG

1

R.LPPLNIGEVLTLPEANFPSFSLPNCN#R.S

IPI00322463

Beta-2-glycoprotein I

1

K.DYRPSAGN#NSLYQDTVVFK.C

IPI00322463

Beta-2-glycoprotein I

1

K.N#ISFACNPGFFLN#GTSSSK.C

IPI00322575

ATP-binding cassette transporter sub-family A member 8a

0.95

K.NTQNILVQN#LSGGQKRK.L

IPI00330747

5730439E10Rik protein

0.94

R.YLMGN#NSSEDSFLTANTVQPLAETGLQLSK.R

IPI00331214

Platelet glycoprotein IV

1

R.QFWIFDVQNPDDVAKN#SSK.I

IPI00331214

Platelet glycoprotein IV

1

K.DPFLSLVPYPISTTVGVFYPYN#DTVDGVYK.V

IPI00331214

Platelet glycoprotein IV

1

K.VISNN#CTSYGVLDIGK.C

IPI00331214

Platelet glycoprotein IV

1

K.RPYIVPILWLN#ETGTIGDEK.A

IPI00331214

Platelet glycoprotein IV

1

K.EN#ITQDPEDH.T

IPI00331214

Platelet glycoprotein IV

1

R.N#LSYWPSYCDMIN#GTDAASFPPFVEK.S

IPI00331259

Desmoglein-1 gamma

1

K.LN#ATDADEPNNLNSMIAFK.I

IPI00331617

Hypothetical olfactomedin-like domain containing protein

1

R.VDKLEEEVSKN#LTK.E

IPI00338565

Mutant fibrillin-1

1

R.VLPFN#VTDYCQLVR.Y

IPI00338785

CDNA, clone:M5C1012G13 product:laminin B1 subunit 1

1

K.MEMPSTPQQLQN#LTEDIR.E

IPI00338785

CDNA, clone:M5C1012G13 product:laminin B1 subunit 1

1

K.QADEDIQGTQNLLTSIESETAASEETLTN#ASQR.I

IPI00338785

CDNA, clone:M5C1012G13 product:laminin B1 subunit 1

1

L.ATGN#VSGGVCDNCQHNTMGR.N

IPI00338785

CDNA, clone:M5C1012G13 product:laminin B1 subunit 1

1

R.VN#ASTTDPN#STVEQSALTR.D

IPI00338785

CDNA, clone:M5C1012G13 product:laminin B1 subunit 1

1

K.LTDTASQSN#STAGELGALQAEAESLDK.T

IPI00339885

Collagen alpha 1(VI) chain

1

R.AALQFLQN#YTVL.A

IPI00339885

Collagen alpha 1(VI) chain

1

L.DDGFLKN#ITAQICIDKK.C

IPI00340463

PREDICTED: similar to hypothetical protein A030003A19

1

K.LLNDYVSN#QTQGMIK.E

IPI00346978

Spink5 protein

0.99

E.TNKNSASRSN#GTGSATGKDVCDQFR.S

IPI00346978

Spink5 protein

0.96

K.GNQDPCMKFQAQMKN#GTLTCPK.G

IPI00348602

Weakly similar to Zinnc finger protein GLI4

0.98

R.FRN#SSNLARHR.R

IPI00350715

PREDICTED: similar to protocadherin 9

1

R.IDPVTGN#ITLEEKPAPTDVGLHR.L

IPI00355606

PREDICTED: expressed sequence AL022779

0.96

M.QN#NSVFGDLK.S

IPI00378430

Ortholog of human Ras association

0.94

R.QETNMAN#FSYR.F

IPI00381122

Weakly similar to Tiarin

1

K.IN#LTTNVVDVNRPLPL.A

IPI00403586

Hypothetical Lipolytic enzymes

1

M.IVNN#HTSLDVER.A

IPI00405742

Plexin B2

0.95

K.QDLALSGN#LSSLYAMTQDK.V

IPI00406434

Mini-agrin

1

K.NELMLN#SSLMR.I

IPI00407222

PREDICTED: similar to human KIAA1815 protein

0.99

H.IPEIN#DTIR.A

IPI00408344

PREDICTED: similar to solute carrier family 4 member 11

0.94

R.EDSLGDEVFDTVN#SSIVSGESIR.F

IPI00409148

Haptoglobin

1

K.NLFLN#HSETASAK.D

IPI00409148

Haptoglobin

1

K.N#LTSPVGVQPILNEHTFCAGLTK.Y

IPI00409148

Haptoglobin

1

K.CVVHYEN#STVPEK.K

IPI00409148

Haptoglobin

1

K.VVLHPN#HSVVDIGLIK.L

IPI00410951

Thyroxine-binding globulin homolog

1

K.VTTCHLPQQN#ATLYK.M

IPI00420489

Von Willebrand factor

1

V.LEGSDEVGEANFN#K.S

IPI00420955

Sortilin 1

1

K.DITNLIN#NTFIR.T

IPI00453607

Killer cell inhibitory receptor-like protein p91A

1

R.LSVLPSPVVTAGGN#MTLH.C

IPI00458917

Sodium/glucose cotransporter 1

1

K.VSNGN#FTAK.E

IPI00459432

Kidney predominant protein

1

S.ADFQGRPVDDPTGAFAN#GSLTFK.V

IPI00460063

Prenylcysteine oxidase

1

K.GELN#STLFSSRPK.D

IPI00461281

NudC domain containing protein 2

1

K.ENPGFDFSGAEISGN#YTK.G

IPI00462999

Ahi-1 isoform III

0.92

D.EFVNTEN#NSSR.K

IPI00463311

PREDICTED: similar to RIKEN cDNA E330026B02

0.99

R.DLGMFAPN#MTR.I

IPI00467180

Translocon-associated protein beta subunit

1

R.IAPASN#VSHTVVLRPLK.A

IPI00467944

61 kDa protein

1

K.VVN#VSELYGTPCTK.R

IPI00468097

340 kDa protein

1

R.NLQVYN#ATSNSLTVK.W

IPI00469000

Zinc transporter SLC39A6

0.98

R.NTNDNIQECFN#TTK.L

IPI00469387

GUGU alpha

1

R.VLYLPAYN#CTLRPVSK.R

IPI00469387

GUGU alpha

1

R.SPPAPPLPQRPLSPLHPLGCN#DSEVLAVAGFALQNINR.D

IPI00469542

Histidine-rich calcium-binding protein

1

R.EVGEEN#VSEEVFR.G

IPI00469839

19 kDa protein

0.91

K.TRTIDVVYN#ASNNELVCTK.T

IPI00471081

RIKEN cDNA 1100001H23

1

K.NGDAYGYYN#DSIK.T

IPI00471273

Apoptosis-related protein 3

1

A.LPEICTLCPGGMHN#LSR.V

IPI00473625

PREDICTED: laminin, alpha 3

0.98

R.FN#ISTPAFQGCMK.N

IPI00473830

Biliary glycoprotein

1

R.MTLSQN#NSILR.I

IPI00475154

Dolichyl-diphosphooligosaccharide-protein glycosyltransferase 63 kDa

1

Q.VLSGCEISVSN#ETK.E

IPI00475157

Serpina1b protein

1

R.ELVHQSN#TSNIFFSPVSIATAFAMLSLGSK.G

IPI00475157

Serpina1b protein

1

N.ASAVFLLPEDGK.M

IPI00551354

PREDICTED: ring finger and KH domain containing 3

0.91

R.N#GSGGGGGGGGGGGGGGSGGGETLDDQR.A

IPI00553278

H-2D cell surface glycoprotein

1

R.NLLGYYN#QSAGGSHTLQQM.S

IPI00554833

Eosinophil-associated ribonuclease 12

1

V.GVCGN#PSGLCSDN#ISQNCHN#SSSR.V

IPI00606550

Ig gamma-2B chain C region, membrane-bound form

1

R.EDYN#STIR.V

IPI00607976

Serine (or cysteine) proteinase inhibitor, clade A, member 3A

1

K.FN#LTETPEADIH.Q

IPI00621319

43 kDa protein

0.92

K.RLFLLDLLN#ATGK.D

IPI00624663

Pzp protein

0.99

K.ACVSLNHVN#ETVM.L

IPI00624761

44 kDa protein

1

R.PVDDPTGAFAN#GSLTFK.V

IPI00626315

38 kDa protein

0.94

P.PSSTDLLWSILN#ASALALLYKTQRDN#ASESK.D

IPI00627061

MKIAA4087 protein

0.94

R.CNIN#GSFSEICHTR.T

IPI00649090

Adult male thymus cDNA, clone:5830446P09 product:CD72 antigen

0.96

V.GSEQPTATWSSVN#SSALRQIPR.C

IPI00649281

52 kDa protein

0.98

R.YHYN#GTLLDGTAFDNSYSR.N

IPI00654271

Myosin light chain, regulatory B

0.91

K.N#PTDAYLDAMMNEAPAPIN#FTMFL.T

IPI00654907

Hypothetical protein CEACAM1/2sec

1

R.FHVHQPVTQPFLQVTN#TTVK.E

P: peptide probability

N#: N-linked glycosylation site

To identify skin tumor-specific proteins, we compared the glycoproteins identified from normal skin, benign, and malignant tumors. Despite the same amount of glycopeptides from each tissue analyzed with the same procedures, the number of unique glycosites identified from different tissues was different. A total of 405 glycosites were identified in cancer tissue, while 252 in benign tissue and 112 in normal skin, when using PeptideProphet score of ≥0.9. The number of glycoproteins identified from papillomas and carcinoma was higher than that of normal tissue. This could be caused by the increased expression of glycoproteins in tumor tissues. A similar observation was also reported from the proteomic analysis of tryptic peptides in a mouse breast cancer model [24].

To determine the glycoprotein changes associated with cancer development, we calculated the relative protein abundance using the number of redundant MS/MS spectra from the same glycoprotein in different tissues [26]. To eliminate the spectral count due to random events, only proteins identified with at least three spectra were included for quantitation. A number of proteins identified in this study were only detected in tumor tissues (benign or malignant) but not in normal tissues (the ratio of such proteins was arbitrary assigned to 100, Table 2). Among the 111 proteins identified with spectral count ratio of at least three folds in cancer or benign tumor tissues comparing to normal tissues, 47 proteins (Table 2) were increased at least three folds in cancer tissues compared to benign tissues. Some of these have been reported to play roles in skin cancer development. These include known extracellular proteins such as thrombospondin, cathepsins, epidermal growth factor receptor, cell adhesion molecules, cadherins, integrins, tuberin, fibulin, and TGFβ receptor. Tenascin-C is an extracellular matrix glycoprotein and plays multiple functions in cell adhesion, migration, growth, and angiogenesis [27, 28]. Tenascin-C has many cell surface receptors, such as integrin and epidermal growth factor receptor, which may affect genome stability associated with interference with genome safeguard functions and escape from cell cycle checkpoints [28]. Tenascin-C has 20 potential N-linked glycosylation sites, but only one glycosylation site (LLQTAEHN#ISGAER, Table 1) has been identified previously (Swiss-Prot Protein knowledgebase, http://us.expasy.org/sprot). In this study, eight N-linked glycosites including the previously identified site were identified in carcinomas (Table 1). They showed increased expression in carcinomas compared to papillomas (Table 2). This observation indicated that Tenascin-C might have increased its glycosylation or abundance during tumor development. In addition, 20 glycoproteins were identified in skin cancer only (Table 2), and these proteins might be used as protein markers to discriminate between the malignant and benign tumors. An example is Arylsulfatase B. In this study, Arylsulfatase B was identified three times only in malignant tissues with two unique glycosylation sites. Arylsulfatase B is a lysosomal enzyme and can degrade proteoglycans in the extracellular matrix and basement membrane. In this way, proteoglycans can obstruct the spread of cancer cells. Therefore, Arylsulfatase B could play a key role in accelerating cancer cell migration [29].
Table 2

Glycoproteins upregulated in skin tumors

IPI

Protein name

Protein location

Ca

Pa

Nr

Total

Ca/Nr

Pa/Nr

Ca/Pa

IPI00119063

AM2 receptor

Transmembrane

17

0

0

17

100.0

0.0

100.0

IPI00381122

Weakly similar to Tiarin

Cell Surface

11

0

0

11

100.0

0.0

100.0

IPI00308971

Cation-independent mannose-6-phosphate receptor

Transmembrane

8

0

0

8

100.0

0.0

100.0

IPI00124265

Latent transforming growth factor beta binding protein 4

Secreted

7

0

0

7

100.0

0.0

100.0

IPI00129304

Collectin sub-family member 12

Transmembrane

7

0

0

7

100.0

0.0

100.0

IPI00129968

Embigin

Transmembrane

7

0

0

7

100.0

0.0

100.0

IPI00153959

Stabilin-1

Transmembrane

5

0

0

5

100.0

0.0

100.0

IPI00316575

Cathepsin K

Cell Surface

4

0

0

4

100.0

0.0

100.0

IPI00321190

Sulfated glycoprotein 1

Secreted

4

0

0

4

100.0

0.0

100.0

IPI00475154

Dolichyl-diphosphooligosaccharide-protein glycosyltransferase 63 kDa

Transmembrane

4

0

0

4

100.0

0.0

100.0

IPI00308785

Prostaglandin G/H synthase 2

Secreted

3

0

0

3

100.0

0.0

100.0

IPI00122737

222 kDa protein

Intracellular

3

0

0

3

100.0

0.0

100.0

IPI00406459

Arylsulfatase B

Secreted

3

0

0

3

100.0

0.0

100.0

IPI00409393

Latent transforming growth factor beta binding protein, isoform 1L

Secreted

3

0

0

3

100.0

0.0

100.0

IPI00119809

Mama protein

Secreted

3

0

0

3

100.0

0.0

100.0

IPI00111960

Lysosomal alpha-glucosidase

Transmembrane

3

0

0

3

100.0

0.0

100.0

IPI00118011

Mannosidase, beta A, lysosomal

Secreted

3

0

0

3

100.0

0.0

100.0

IPI00121120

Procollagen, type V, alpha 2

Secreted

3

0

0

3

100.0

0.0

100.0

IPI00129158

Tyrosine-protein phosphatase non-receptor type substrate 1

Transmembrane

3

0

0

3

100.0

0.0

100.0

IPI00120769

Solute carrier family 29 (nucleoside transporters), member 1

Transmembrane

5

0

1

6

5.0

0.0

100.0

IPI00415773

Integrin alpha-M

Transmembrane

34

3

1

38

34.0

3.0

11.3

IPI00338785

cDNA, clone:M5C1012G13 product:laminin B1 subunit 1

Intracellular

9

1

3

13

3.0

0.3

9.0

IPI00130627

Legumain

Secreted

17

2

0

19

100.0

100.0

8.5

IPI00113480

Myeloperoxidase

Secreted

8

1

0

9

100.0

100.0

8.0

IPI00113824

Basement membrane-specific heparan sulfate proteoglycan core protein

Cell Surface

15

2

5

22

3.0

0.4

7.5

IPI00124830

Leukocyte surface antigen CD47

Transmembrane

7

1

1

9

7.0

1.0

7.0

IPI00320605

Integrin beta-2

Transmembrane

20

3

0

23

100.0

100.0

6.7

IPI00308990

Monocyte differentiation antigen CD14

Cell Surface

13

2

0

15

100.0

100.0

6.5

IPI00133082

CD177 antigen

Secreted

6

1

0

7

100.0

100.0

6.0

IPI00130486

FK506-binding protein 9

Cell Surface

5

1

0

6

100.0

100.0

5.0

IPI00308609

VESICULAR INTEGRAL-MEMBRANE PROTEIN VIP36

Transmembrane

5

1

0

6

100.0

100.0

5.0

IPI00403938

Tenascin-C

Cell Surface

133

29

0

162

100.0

100.0

4.6

IPI00462199

Basigin

Transmembrane

13

3

0

16

100.0

100.0

4.3

IPI00120245

Integrin alpha-V

Transmembrane

8

2

0

10

100.0

100.0

4.0

IPI00120245

Integrin alpha-V

Transmembrane

8

2

0

10

100.0

100.0

4.0

IPI00110852

Translocon-associated protein alpha, muscle specific isoform

Cell Surface

4

1

0

5

100.0

100.0

4.0

IPI00125266

Acid ceramidase

Secreted

4

1

0

5

100.0

100.0

4.0

IPI00121038

Versican core protein

Cell Surface

11

3

2

16

5.5

1.5

3.7

IPI00124283

Macrophage scavenger receptor types I and II

Transmembrane

7

2

0

9

100.0

100.0

3.5

IPI00132067

Fibulin-2

Secreted

31

9

0

40

100.0

100.0

3.4

IPI00223769

CD44 antigen

Transmembrane

10

3

0

13

100.0

100.0

3.3

IPI00127447

Lysosome membrane protein II

Transmembrane

32

10

0

42

100.0

100.0

3.2

IPI00322447

RA175

Transmembrane

6

2

0

8

100.0

100.0

3.0

IPI00154057

Protocadherin 1

Cell Surface

3

1

0

4

100.0

100.0

3.0

IPI00121312

MFIRE1

Secreted

3

1

0

4

100.0

100.0

3.0

IPI00124640

Osteoclast-like cell cDNA, clone:I420031M06 product:granulin

Secreted

3

1

0

4

100.0

100.0

3.0

IPI00134483

Lectin lambda

Cell Surface

3

1

1

5

3.0

1.0

3.0

IPI00272381

Proline 4-hydroxylase, alpha 1

Secreted

17

6

0

23

100.0

100.0

2.8

IPI00122493

FK506-binding protein 10

Secreted

7

3

0

10

100.0

100.0

2.3

IPI00123831

SDR1 protein

Transmembrane

11

5

0

16

100.0

100.0

2.2

IPI00224728

Cd63 antigen

Transmembrane

8

4

0

12

100.0

100.0

2.0

IPI00128689

Collagen alpha 1(V) chain

Secreted

6

3

0

9

100.0

100.0

2.0

IPI00125877

Hypothetical protein

Transmembrane

6

3

0

9

100.0

100.0

2.0

IPI00130015

Dipeptidyl-peptidase I

Secreted

4

2

0

6

100.0

100.0

2.0

IPI00318012

T-cell immunomodulatory protein

Transmembrane

4

2

0

6

100.0

100.0

2.0

IPI00123678

Cadherin-22

Transmembrane

2

1

0

3

100.0

100.0

2.0

IPI00126316

Mast cell carboxypeptidase A

Secreted

2

1

0

3

100.0

100.0

2.0

IPI00130661

Tripeptidyl-peptidase I

Cell Surface

2

1

0

3

100.0

100.0

2.0

IPI00131366

Keratin, type II cytoskeletal 6B

Transmembrane

2

1

0

3

100.0

100.0

2.0

IPI00221418

hypothetical Phospholipase D/Transphosphatidylase

Transmembrane

2

1

0

3

100.0

100.0

2.0

IPI00279051

RIKEN cDNA A930025J12

Transmembrane

2

1

0

3

100.0

100.0

2.0

IPI00554833

Eosinophil-associated ribonuclease 12

Secreted

2

1

0

3

100.0

100.0

2.0

IPI00127280

Myeloid bactenecin

Secreted

43

22

0

65

100.0

100.0

2.0

IPI00118413

Thrombospondin 1

Secreted

20

11

0

31

100.0

100.0

1.8

IPI00127352

AMBP protein

Secreted

22

14

0

36

100.0

100.0

1.6

IPI00132600

Niemann-Pick C1 protein

Transmembrane

3

2

0

5

100.0

100.0

1.5

IPI00137177

Lysosomal protective protein

Secreted

3

2

0

5

100.0

100.0

1.5

IPI00132474

Integrin beta-1

Transmembrane

18

13

1

32

18.0

13.0

1.4

IPI00123342

Hypoxia up-regulated 1

Secreted

19

14

0

33

100.0

100.0

1.4

IPI00126090

Integrin alpha-3

Transmembrane

4

3

0

7

100.0

100.0

1.3

IPI00131881

ADAM 10

Cell Surface

4

3

0

7

100.0

100.0

1.3

IPI00406434

Mini-agrin

Secreted

4

3

0

7

100.0

100.0

1.3

IPI00410951

Thyroxine-binding globulin homolog

Secreted

4

3

0

7

100.0

100.0

1.3

IPI00125058

Laminin alpha-3 chain

Secreted

9

7

1

17

9.0

7.0

1.3

IPI00112326

Epithelial membrane protein 1

Transmembrane

6

5

0

11

100.0

100.0

1.2

IPI00128154

Cathepsin L

Secreted

23

20

0

43

100.0

100.0

1.2

IPI00121362

F11r protein

Transmembrane

9

9

0

18

100.0

100.0

1.0

IPI00108535

Carcinoembryonic antigen-related cell adhesion molecule 1

Cell Surface

7

7

0

14

100.0

100.0

1.0

IPI00407222

PREDICTED: similar to human KIAA1815 protein

Transmembrane

6

6

0

12

100.0

100.0

1.0

IPI00128989

Vacuolar ATP synthase subunit S1

Transmembrane

5

5

0

10

100.0

100.0

1.0

IPI00471081

RIKEN cDNA 1100001H23

Cell Surface

5

5

0

10

100.0

100.0

1.0

IPI00226932

Quinoprotein alcohol dehydrogenase structure containing protein

Secreted

4

4

0

8

100.0

100.0

1.0

IPI00127672

PREDICTED: hypothetical protein LOC66967

Secreted

2

2

0

4

100.0

100.0

1.0

IPI00346978

Spink5 protein

Secreted

2

2

0

4

100.0

100.0

1.0

IPI00469387

GUGU alpha

Secreted

23

23

3

49

7.7

7.7

1.0

IPI00134549

Lysosome-associated membrane glycoprotein 2

Transmembrane

8

9

0

17

100.0

100.0

0.9

IPI00121430

Collagen alpha 1(XII) chain

Secreted

11

14

0

25

100.0

100.0

0.8

IPI00122272

Extracellular matrix protein 1

Secreted

11

14

0

25

100.0

100.0

0.8

IPI00227969

Integrin alpha-6

Transmembrane

6

8

1

15

6.0

8.0

0.8

IPI00134652

Type VII collagen

Secreted

5

7

0

12

100.0

100.0

0.7

IPI00114256

Synaptophysin-like protein

Transmembrane

10

14

3

27

3.3

4.7

0.7

IPI00110810

Prostate stem cell antigen

Secreted

9

13

0

22

100.0

100.0

0.7

IPI00467180

Translocon-associated protein beta subunit

Transmembrane

15

22

0

37

100.0

100.0

0.7

IPI00133172

Serpin B11

Intracellular

2

3

0

5

100.0

100.0

0.7

IPI00111013

Cathepsin D

Secreted

19

30

0

49

100.0

100.0

0.6

IPI00117093

Laminin beta-3 chain

Cell Surface

3

6

0

9

100.0

100.0

0.5

IPI00130342

Lymphocyte antigen 6 complex locus G6C protein

Secreted

2

4

0

6

100.0

100.0

0.5

IPI00125293

Eosinophil cationic protein 1

Secreted

1

2

0

3

100.0

100.0

0.5

IPI00320204

RIKEN cDNA 2210023G05

Secreted

1

2

0

3

100.0

100.0

0.5

IPI00468097

340-kDa protein

Secreted

4

8

1

13

4.0

8.0

0.5

IPI00113853

Desmocollin-3

Transmembrane

2

6

0

8

100.0

100.0

0.3

IPI00319814

Suprabasal-specific protein suprabasin

Secreted

3

10

0

13

100.0

100.0

0.3

IPI00115854

TROP2 protein

Transmembrane

1

4

0

5

100.0

100.0

0.3

IPI00127933

Androgen binding protein alpha

Secreted

1

4

0

5

100.0

100.0

0.3

IPI00130249

GPI-anchored metastasis-associated protein homolog

Secreted

10

60

0

70

100.0

100.0

0.2

IPI00111014

Elongation of very long chain fatty acids protein 4

Transmembrane

3

20

2

25

1.5

10.0

0.2

IPI00129243

Gamma-glutamyl hydrolase

Secreted

1

8

0

9

100.0

100.0

0.1

IPI00338790

Glandular kallikrein KLK13

Cell Surface

0

6

0

6

0.0

100.0

0.0

IPI00111115

Similar to METASTASIS-ASSOCIATED GPI- ANCHORED PROTEIN

Secreted

0

4

0

4

0.0

100.0

0.0

IPI00473830

Biliary glycoprotein

Transmembrane

0

4

0

4

0.0

100.0

0.0

IPI00153548

Hypothetical protein

Transmembrane

0

3

0

3

0.0

100.0

0.0

Ca/Nr: Ratio of spectral count of carcinomas to normal tissue, Pa/Nr: Ratio of spectral count of papillomas to normal tissue, Ca/Pa: Ratio of spectral count of carcinomas to papillomas

Here, we determined the relative abundance of glycosylated proteins using identified glycosylated peptides. However, glycosylation for individual glycosites from the same protein might be different and can be determined by quantitative analysis of each glycosite. In addition, changes in glycan structure that may be important to the disease cannot be determined by this method, and specific enrichment of glycopeptides with certain glycan structure is needed.

Detected Tissue-Derived Proteins in Plasma

Since the plasma proteome is dominated by several highly abundant proteins, proteins released from specific tissues would normally be present at low abundance in plasma, and their detection might be obscured by the highly abundant plasma proteins. To detect tumor-specific proteins in plasma, we used isotopic labeling to detect the isotopic peaks that consisted of the tissue-derived proteins from both plasma and tissues.

The glycopeptides from four carcinomas were labeled with d413C4-succinic anhydride. The glycopeptides from plasma of the four mice before and after cancer development were labeled with d013C0 and d413C0-succinic anhydride, respectively. To monitor the labeling efficiency, we spiked the same amount of standard peptide from angiotensin (0.1 μg) in the glycopeptides isolated from carcinomas and plasma samples as labeling control. Then, all the labeled peptides were combined for MS analysis. The mixture was separated by 2D Nano-LC then analyzed by MALDI-TOF/TOF. Free angiotensin (ms 1296.68) was not observed after labeling. Instead, 100, 104, and 108 Da shifted from 1296.68 were observed in equal amounts in the mixed samples. This indicates the efficient and quantitative isotopic labeling using succinic anhydride.

The mixed glycopeptides from carcinomas and plasma samples contained both skin-cancer-related peptides and peptides from plasma. In order to detect glycopeptides associated with skin cancer in plasma, we focused our analysis on glycopeptides previously identified as cancer-associated glycoproteins from skin tumors in the mixture (Table 2) and avoided the analysis of plasma proteins. To achieve this goal, the peptide peaks that contained masses from glycopeptides specifically identified from carcinomas and their isotopic pairs from plasma were selected for MS/MS analysis.

Two types of paired patterns were observed. One was that the intensity of d413C4-labeled peptides (with 8 mass unit shift for each amino group from peptides derived from cancer tissues) was much greater than d413C0-labeled peptide (with 4 mass unit shift for each amino group from peptides derived from plasma of cancer-bearing mice), and intensity of d013C0-labeled peptide (with 0 mass unit shift for each amino group from peptides derived from plasma before carcinogen induction) was lower than that of peptides from plasma of cancer-bearing mice. This pattern indicated that the peptide was from tumor-specific protein and detectable in cancer plasma at low intensity. The other pattern was that similar or lower intensity of peptides from cancer tissues than in plasma, and peptides with this pattern were derived from plasma proteins.

Tumor-associated glycopeptides could be detected in plasma. Tenascin-C was identified in carcinomas with 133 spectra, and it was also identified in benign papillomas with 29 spectra. However, none of these glycopeptides were identified in normal tissue (Table 2). In plasma, the labeled peptide peak of Tenascin-C from cancer was found with its paired peak from cancer plasma (Fig. 2A), which indicated that it was also detected in plasma after cancer development, but not in control plasma before the carcinogen treatment. Another skin tumor-specific glycoprotein, Arylsulfatase B, was also detected in plasma successfully in a similar way (Fig. 2b). These data indicated that extracellular proteins associated with tumor development were identifiable in plasma from tumor-bearing mice using glycopeptide capture, isotopic labeling, and mass spectrometry.
Fig. 2

Detection of tumor-specific proteins in plasma. a The detected paired peaks of succinic-anhydride-labeled Tenascin-C and MS/MS spectrum of Tenascin-C. b The paired peak of succinic-anhydride-labeled Arylsulfatase B and MS/MS of Arylsulfatase B. c The paired peak of succinic-anhydride-labeled Ig gamma-3 chain C region showed different peak pattern from Tenascin-C and MS/MS of Ig gamma-3 chain C region. NrP mouse plasma without carcinogen treatment, CaP mouse plasma from cancer-bearing mice after carcinogen treatment, MT mouse cancer tissues

One of the advantages of using this tissue-targeted approach is that tumor-associated proteins can be identified in plasma even if they are present in very low abundance. The peptides from cancer tissue are likely to be at higher abundance compared to the same peptides in plasma. This allowed us to determine their masses and peptide sequences in the mixture using isotopic peaks from tumors. Using this information, tumor-derived peptides in plasma can be identified, while they are not identifiable by data-dependent MS/MS acquisition and database searches. Both Tenascin-C and Arylsulfatase B are low abundant proteins. They were not identified in plasma before cancer development, and their detection in plasma was associated with cancer development.

Proteins from plasma can also be detected in tissues and plasma as isotopic pairs due to vascularization of the tissue. If a glycopeptide detected in both cancer tissues and plasma was derived from plasma, the peptide peak showed similar or lower intensity in cancer tissues than that in plasma. An example of this was the identification and quantification of glycoprotein, Ig gamma-3 chain C region, in tissue and plasma. However, its paired peptide peaks were found in a different pattern from that observed with Tenascin-C (Fig. 2c). The intensities of d013C0- and d413C0-labeled peptides from plasma before and after tumor induction were much higher than that from d413C4-labeled peptides from tumors. This indicated that this peptide was from a plasma-derived protein, and Ig gamma-3 could be detected from tissue due to the blood contamination in the tumor.

The methodology of targeted detection of tumor proteins using glycopeptide capture, isotopic labeling, and mass spectrometry is based on the analysis of N-linked glycopeptides to study extracellular proteins from tumors and plasma. It has been shown to increase the detectability of tumor proteins by focusing on the same subset of glycopeptides in both tumors and plasma [13]. The tumor-associated glycopeptides could be detected in plasma on account of the several advantages of our methodologies. First, the glycopeptide capture method dramatically reduces the sample complexity. Non-glycoproteins and non-glycopeptides from glycoproteins were removed from the pool of samples. For example, albumin, the most abundant serum protein, was automatically transparent to this method since it does not contain N-linked glycosylation. Second, the glycopeptide isolation method could be used to enrich extracellular proteins due to the fact that most extracellular proteins are glycosylated and likely to enter the bloodstream. Third, we used an isotopic labeling method to facilitate the detection of tumor proteins within complex plasma by identifying paired peptide peaks from tumor tissues and plasma. However, the method described here is only for proteins that contain N-linked glycosylation. For proteins that do not contain N-linked glycosylation, this method will miss the detection of those proteins.

These results show that our strategy for detection of tumor-specific proteins in plasma is specific and sensitive for low abundant tumor-associated proteins. Different from the previous report of identification of prostate cancer-derived proteins in serum using xenograft-bearing mice [30], our study is more focused on tumor-associated extracellular proteins that are likely to be used in early detection.

Conclusions

In this study, we described a platform for quantitative detection of tumor-specific extracellular proteins derived from tumors and plasma. The fact that tumor-specific proteins were detectable in plasma from tumor-bearing mice indicates that cancer-specific markers could be detected in plasma using targeted approaches, and these proteins could be serum tumor marker candidates [7]. Once such candidate proteins are identified, the homologues of the proteins can be verified in human sera using the targeted approach. Enzyme-linked immunosorbent assays can be developed using a pair of antibodies. However, if antibodies against the candidate proteins are not available, mass-spectrometry-based methods can be applied to detect candidate proteins in plasma. One approach is referred to as MRM [1719]. In another approach, called stable isotope standards and capture by anti-peptide antibodies, a specific peptide from sample and the synthetic heavy isotope-labeled peptide of the candidate protein are captured by peptide antibody. The mass spectrometer is then used to detect and quantify the specific peptide with known precursor mass and fragmentation ions [31].

Declarations

Acknowledgment

This work was supported with federal funds from the National Cancer Institute, National Institutes of Health, by Grants R21-CA-114852. We gratefully acknowledge the support from the Mass Spectrometry Facility at the Johns Hopkins University and the support of Trans-Proteomic Pipeline (TPP) Software tools available from the Aebersold Group at the Institute for Systems Biology.

Authors’ Affiliations

(1)
Department of Pathology, Johns Hopkins University
(2)
Human Biology and Public Health Science, Fred Hutchinson Cancer Research Center
(3)
Department of Pathology, Johns Hopkins University

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Copyright

© Humana Press 2008