Expression of chloride intracellular channel protein 1 (CLIC1) and tumor protein D52 (TPD52) as potential biomarkers for colorectal cancer

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Clinical Biochemistry 41 (2008) 1224 – 1236

Expression of chloride intracellular channel protein 1 (CLIC1) and tumor protein D52 (TPD52) as potential biomarkers for colorectal cancer Darinka Todorova Petrova a,b , Abdul R. Asif b , Victor W. Armstrong b , Ivanka Dimova a , Svetoslav Toshev c , Nikolay Yaramov c , Michael Oellerich b , Draga Toncheva a,⁎ b

a Department of Medical Genetics, Medical University, Sofia, Bulgaria Department of Clinical Chemistry, University of Medicine, Goettingen, Germany c Department of Surgery, Aleksandrovska University Hospital, Sofia, Bulgaria

Received 28 March 2008; received in revised form 17 July 2008; accepted 18 July 2008 Available online 30 July 2008

Abstract Objectives: Unequivocal biomarkers are needed to predict susceptibility and progression of colorectal cancer. Design and methods: Paired samples of tumor and normal tissue from six patients with colorectal cancer of different localization, pTNM stage and grade were employed in the present study. MS analysis was used to identify differentially regulated proteins after 2-DE separation and densitometric analysis. Results: Densitometric analysis revealed differential abundance of 55 spots in tumor as compared to normal tissues. Thirty nine out of 55 spots were unambiguously identified by MS representing 32 different proteins. CLIC1, TPD52 and FABPL were consistently overexpressed (N 3-fold, P b 0.05) in all tumor tissue samples, while TPM1, TPM2, TPM3, TAGL and MLRN were consistently down-regulated (N3-fold, P b 0.05) compared to normal tissue. Conclusions: CLIC1 and TPD52 were significantly (P b 0.05) up-regulated in all cases of colorectal cancer investigated, irrespective of localization, pTNM stage and grade of colon cancer highlighting their potential to serve as new biomarkers. © 2008 The Canadian Society of Clinical Chemist. Published by Elsevier Inc. All rights reserved. Keywords: Proteomics; colorectal cancer; CLIC1; TPD52; ANXA5

Introduction Colon cancer continues to rank as a serious public health concern [1]. Transformation of normal cells into malignant cells is considered to be a multi-step process in colorectal carcinogenesis involving loss of function of tumor suppressor genes, as well as activation of oncogenes [2,3]. Transformation can be caused by genetic changes leading to essential alterations or by gain of function [4,5]. Candidate biomarkers are needed for early detection and prediction of the cancer. Proteome studies of colorectal cancer have previously been reported by several groups [1,6–11] demonstrating a number of ⁎ Corresponding author. Department of Medical Genetics, Genomic Centre for Common Diseases, Medical University-Sofia, 2 Zdrave str., 1431 Sofia, Bulgaria. Fax: +00359 2 9520357. E-mail address: [email protected] (D. Toncheva).

proteins with altered expression levels. Despite these efforts, availability of reliable tumor markers still remains a major challenge. Our study aimed to detect tumor-specific changes in the proteome of human colorectal cancers by comparing the normal and tumor tissue from the same patient. Materials and methods Sample preparation Six unrelated Bulgarian patients with newly diagnosed sporadic colorectal cancer participated in this study. The cancer was diagnosed by histopathological examination using established clinical criteria [12,13]. The patients were anonymised according to ethical and legal standards. Patients' medical characteristics are presented in Table 1. Tumor and neighboring normal intestinal tissues were collected from each patient at

0009-9120/$ - see front matter © 2008 The Canadian Society of Clinical Chemist. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2008.07.012

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Table 1 Demographic data of the donors (each normal and tumor tissue was obtained from the same individual) Patient, no.

1

2

3

4

5

6

Gender Age, years Tumor localization pTNM Grade Sport/week Vegetarian Cigarettes/day Alcohol (wine)/day Other diseases

Male 63 Rectum pT2N0M0 G2 No No 20 100–200 mL Hypertension

Female 70 Coecum pT2N0M0 G2 No No No No No

Female 80 Sigmoid colon pT2N0M0 G1 No No No 300 mL Hypertension

Male 45 Sigmoid colon pT3N0M0 G2 Riding a bicycle 2 times No 20 100 mL Gastric ulcer

Female 68 Rectum pT3N1M1 G3 No No No No Hypertension

Female 62 Flexura coli hepatica pT4N1M1 G3 No No Not now but in the past 10 No No

surgery at the Aleksandrovska University Hospital, Sofia, Bulgaria according to the protocol approved by its Ethics Committee. The neighboring normal tissue obtained at ca. 15 cm distance from the tumor margin was histologically determined as normal (healthy) mucosa. Total protein was isolated by homogenization (Polytron® PT 3100, Kinematica AG, Kriens, Switzerland) and sonification (Branson sonifier 250, Branson, USA). Protein concentration was determined according to the Bradford method [14] using the Bio-Rad protein assay kit (Bio-Rad, Munich, Germany). Aliquots from the samples were stored at − 80 °C. Two-dimensional electrophoresis (2-DE) Two representative 2-DE gels per tissue sample were produced according to the protocol of Gorg et al. [15]. Protein samples, 125 μg protein/17-cm immobilized pH gradient (IPG) strip with a linear pH range 3–10 (ReadyStrip™, Bio-Rad, Munich, Germany), were passively rehydrated. Isoelectric focusing was performed in a Protein IEF Cell (Bio-Rad, Munich, Germany). After equilibration each strip was loaded onto a vertical 12.5% polyacrylamide SDS PAGE. The gels were stained according to the modified silver staining method of Blum et al. [16], scanned (CanoScan 8400F, Canon, Germany) and finally dried (Gel Dryer, Model 583, Bio-Rad, Munich, Germany). Spot quantification was done using a Delta2D version 3.4 (DECODON GmbH, Greifswald, Germany) [17]. The gel images were normalized to the total spot volume of each gel. Nonparametric analysis (Wilcoxon test) using SPSS, version 14.0 for Windows was performed to evaluate the significant differences in protein expression between tumor and normal tissues. Mass spectrometric analysis In-gel digestion with trypsin (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was carried out according to a modified procedure described previously [18]. Peptide sequences were determined using a Q-TOF Ultima Global (Micromass, Manchester, UK) mass spectrometer equipped with a nanoflow ESI Z-spray source in positive ion mode as published elsewhere [19,20]. Processed data were searched against SwissProt database through the Mascot search algorithm with oxidation (M) and carbamidomethyl (C) modification when appropriate.

Western blotting Protein extract (20 μg) from each sample was loaded onto a 12.5% SDS-PAA vertical minigel slot (Mini-Protean II electrophoresis chamber, Bio-Rad, Munich, Germany). After the electrophoresis the proteins were electrotransfered to nitrocellulose membranes (Protran BA 85 0.45 μm, Schleicher & Schuell, Dassel, Germany). Ponceau red staining was used to ensure adequate transfer. The membranes were incubated with a tumor protein D52 (TPD52) mouse polyclonal antibody (Catalog ID H00007163-A01, Abnova Corporation, Taipei, Taiwan), or with an annexin A5 (ANXA5) rabbit polyclonal antibody (Catalog ID NB100-1930, Abcam®, Acris antibodies, Hiddenhausen, Germany) and then with goat anti-mouse IgG, whole molecule (Catalog ID A3562, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) or goat anti-rabbit IgG, H+L (Catalog ID 170-6518, Bio-Rad Laboratories, Hercules, CA). The blots were stained using an AP conjugate substrate kit (BioRad, Munich, Germany) and scanned (CanoScan 8400F, Canon, Germany). Band intensities were analyzed by the LabImage software (LabImage version 27.1, Kapelan GmbH, Halle, Germany) and Wilcoxon test. Results Silver-stained 2-DE gels were obtained from paired tumor and adjacent healthy non-tumor tissue samples from six patients with colorectal cancer (Fig. 1). The normal and tumor tissues were obtained from the same individuals and the variability of their demographic data and protein expression are shown in Table 1 and Appendix A. On comparative computational analysis of the images, 55 spots revealed changes in the relative intensity (% volume) with an average ratio of either b 0.6 or N1.5 (tumor material versus healthy tissue). Thirty nine out of the 55 analyzed spots could be unambiguously identified by mass spectrometry, which represented 32 different proteins (Fig. 2, Table 2). Significant differences (P b 0.05) in protein expression were observed between tumor and normal tissue for most of the proteins (Appendix A). The matching sequenced peptides along with their scores are listed in Appendix B. Twenty-nine of these proteins were only identified through a single spot. Three proteins, however, displayed two or more variant forms. These proteins were fatty acid-binding protein, liver (FABPL, spot labels 254 and 255), transgelin (TAGL, spot

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Fig. 1. 2-DE gels of tumor (a) and adjacent normal (b) colorectal tissue from the same patient.

labels 202, 215, 219, 222, 223, and 231) and destrin (DEST, spot labels 235 and 238) (Fig. 1, Table 2, Appendix A). Chloride intracellular channel protein 1 (CLIC1, spot label 146, average T/N = 3.04), TPD52 (spot label 179, average T/ N = 3.64), and FABPL (spot labels 254 and 255, average T/ N = 4.07 and 3.08) (Table 2, Appendix A) showed significant

up-regulation in all tumor tissues investigated, with a mean ratio N 3.0 (P b 0.05) compared to paired normal tissue. Tropomyosin-1 alpha chain (TPM1, spot label 94 RU, average T/N = 0.31), tropomyosin beta chain (TPM2, spot label 94 L, average T/N = 0.21), tropomyosin alpha-3 chain (TPM3, spot label 94 RO, average T/N = 0.31), TAGL (spot labels 202,

Fig. 2. Mean spot intensity of 39 differentially regulated spots from 6 patients with colorectal cancer (6 tumor and 6 normal tissues). The bar chart illustrates those spots with a mean intensity ratio (tumor versus normal tissue) of either b0.6 or N1.5.

D.T. Petrova et al. / Clinical Biochemistry 41 (2008) 1224–1236

219, 222, 231, resp. average T/N = 0.28, 0.21, 0.21, 0.31), and myosin regulatory light chain 2, smooth muscle isoform (MLRN, spot label 224, average T/N = 0.26) were significantly downregulated in all tumor samples with a mean ratio b0.33 (P b 0.05) compared to paired normal tissue (Table 2, Appendix A). The expression levels of two of the regulated proteins [TPD52 (spot label 179, average T/N = 3.64, P b 0.05) and ANXA5 (spot label 132, average T/N = 1.9, P b 0.05)] were further confirmed by Western blotting (Fig. 3). A specific band of ca. 26 kDa was observed in all extracts using a mouse polyclonal antibody raised against partial recombinant TPD52 for Western blotting (Fig. 3a). Consistent up-regulation was detected in all tumor samples (2.05-fold, P b 0.05). Its molecular weight agreed with that reported previously, 24–27 kDa [21,22] and with the observed molecular weight in our 2-DE gels (ca. 27 kDa). A protein band of ca. 33 kDa was observed in all tested patients using a rabbit polyclonal antibody against ANXA5 (Fig. 3b). However, up-regulation (P N 0.05) was detected only in the two patients with rectal cancer (2.32-fold). The molecular weight agreed with the calculated (ca. 36 kDa) and the observed (ca. 34 kDa) mass in our 2-DE gels. Discussion We analyzed tumor and neighboring normal tissue from six patients with different tumor localization, pTNM stage and grade of colon cancer and found 32 differentially expressed proteins (Table 2). Several proteins have been previously reported in other studies using 2-DE to be associated with colorectal cancer (Table 2). The observed protein expression in the present study confirmed the results of previous studies for the following proteins: up-regulation in tumor of KPYM [10,11], elongation factor 1-gamma (EF1G) [1], inorganic pyrophosphatase (IPYR) [1,10], VDAC1 [10], annexin A3 (ANXA3) [1], annexin A4 (ANXA4) [1,6,9], CLIC1 [10], oncogene DJ1 (PARK7) [1] and PRDX5 [10], and downregulation of desmin (DESM) [1,8] and TPM2 [10]. Previous studies with colorectal carcinoma samples have, however, reported contradictory results for several proteins: ANXA5 [1,6], FABPL [8,9], VIME [1,6,8,10,11], TPM1 [1,10], TAGL [1,7]. We presume that these discrepancies are due to different demographic data of the patients, tumor localization and/or different criteria for collection and preparation of the samples. We found several significantly (P b 0.05) regulated proteins in colorectal cancer, which were not described by other authors who also used 2-DE and mass spectrometry (Table 2, Appendix A). These proteins include regulatory or signaling molecules [glucosidase 2 subunit beta precursor (GLU2B), electron transfer flavoprotein subunit alpha, mitochondrial precursor (EFTU), glyoxalase domain-containing protein 4 (GLOD4), TPD52], proteins involved in hydrolization [abhydrolase domain-containing protein 14B (AB14B), acyl-protein thioesterase 1 (LYPA1)], in keratinocyte differentiation [fatty acidbinding protein, epidermal (FABPE)], in electron acceptance (ETFA), in oxidoreduction [superoxide dismutase (SODC)], as well as in cell adhesion, contraction and the cytoskeleton

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[epithelial-cadherin precursor (CADH1), TPM3, MLRN, cofilin-2 (COF2), DEST, myosin light polypeptide 6 (MYL6), profilin-1 (PROF1)]. Recently, CLIC1 was proposed as a novel potential prognostic and therapeutic agent in gastric carcinoma, which was significantly up-regulated in 67.9% of the studied patients [23]. CLIC1 expression was elevated 1.95-fold in gastric, 1.8fold in cervical, 3.5-fold in liver [23], 3.3-fold in breast ductal [24], and 1.98–3.04-fold in colorectal cancer [resp. 10, present study]. Elevated CLIC1 expression was strongly correlated with lymph node metastasis, lymphatic and perineural invasion, pathological staging, and poor survival [23]. It was presumed that CLIC1 overexpression modulates cell division and/or antiapoptosis signaling, resulting in cellular transformation [25]. Our 2-DE analysis revealed consistent up-regulation in tumor samples of TPD52 (P b 0.05), which was further confirmed by Western blot findings (Fig. 3a). It was reported that TPD52 represents an early tumor marker in ovarian [26] and prostate cancer [27,28]. TPD52 can induce immunoglobulin G (IgG) antibodies in human breast cancer which suggests a lack of normal immunologic tolerance to TPD52, and TPD52 may be sufficiently immunogenic to be explored as an anticancer vaccine [29]. Lewis et al. [22] demonstrated that expression of the murine orthologue of human TPD52 (mD52) is critical for lung tumorigenesis and progression to metastasis. They reported increased TGF-β1 expression and secretion and a decreased expression of its receptor (TGF-βR1/ALK-5) in 3T3.mD52 cells. The increased secretion of TGF-β1 is a common event in malignant cells. It activates proteases that degrade the extracellular matrix thereby enabling invasion and metastasis [30,31]. Increased TGF-β1 and decreased TGF-βR1 levels in the tumor microenvironment lead to increased cancer risk and metastasis in several human cancers i.e., cancers of the kidney and bladder [32], colon [33], pancreas [34] and prostate [35]. Annexin family members regulate processes such as ion flux, endocytosis, intracellular vesicular transport, exocytosis, cellular adhesion, calcium sensors, and signal transduction. During apoptosis, phosphatidylserine is translocated from the cytoplasmic face of the plasma membrane to the cell surface. One member of the annexin family, ANXA5, has a high Ca2 + -dependent affinity for phosphatidylserine and therefore is used as a probe for detecting apoptosis. Although the results from 2-DE gels showed higher expression of ANXA5 in all tumor tissues of the present study, its expression was confirmed only for rectal cancer, but not for other cancer localizations using Western blot analysis (Fig. 3b). One possible explanation of this finding may be the use of polyclonal antibodies with lower specificity in the present investigation. In conclusion, 32 different proteins exhibited differential expression in tumor tissues compared to non-tumor tissues from the same patient with colorectal cancer. Sixteen of 32 proteins were newly identified by 2-DE and mass spectrometry. These proteins have been shown to act in cell regulation, apoptosis, keratinocyte differentiation, oxidoreduction, as well as cell

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Table 2 Differentially expressed proteins identified using 2-DE and mass spectrometry Spot label

Protein name

4

Average T/N

Acc. no.

Mr, kDa Calc./Obs.

–

P14314

59.388/∼ 100 4.33/∼ 4.5

Regulatory subunit of glucosidase II.

2.41

–

P12830

97.396/∼ 95

4.58/∼ 4.2

Pyruvate kinase isozyme M1/M2 (KPYM)

2.30

Negative in normals P14618 [11]; 5.77 [10]

57.900/∼ 60

7.96/∼ 9.1

Elongation factor 1-gamma (EF1G) Elongation factor Tu, mitochondrial precursor (EFTU)

2.28

1.49 [1]

P26641

49.956/∼ 48

6.25/∼ 7

Calcium-dependent protein involved in regulating cell–cell adhesions, mobility and proliferation of epithelial cells. Glycolytic enzyme catalyzing transfer of a phosphoryl group from phosphoenolpyruvate to ADP, generating ATP. Nucleic acid-binding protein.

1.92

–

P49411

49.510/∼ 46

125

Inorganic pyrophosphatase (IPYR)

3.95

1.99 [1]; 3.11 [10]

Q15181

32.639/∼ 38

131

Voltage-dependent anionselective channel protein 1 (VDAC1) Annexin A5 (ANXA5)

2.42

6.87 [10]

P21796

30.623/∼ 30

7.26/∼ 7.25 Nucleic acid-binding protein, promoter of GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein biosynthesis. 5.54/∼ 6 Magnesium binding hydrolase, important for the phosphate metabolism of cells. 8.62/∼ 9.5 Mitochondrial membrane protein porin, involved in apoptosis.

1.90

0.33 [6]; 2.76, 1.83 [1]

P08758

35.783/∼ 34

Present study

Previous studies

Glucosidase 2 subunit beta precursor (GLU2B) Epithelial-cadherin precursor (CADH1)

2.43

36

53 L

9

74

132

pI Calc./Obs.

4.94/∼ 5.2

1.61

–

Q9HC38 34.771/∼ 35

5.4/∼ 6

136 O

Glyoxalase domaincontaining protein 4 (GLOD4) Annexin A3 (ANXA3)

2.12

3.04 [1]

P12429

36.353/∼ 36

5.63/∼ 6.1

136 U

Annexin A4 (ANXA4)

1.78

3.4 [6]; 2.3 [1]; 2.32 [9]

P09525

35.860/∼ 35

140

Electron transfer flavoprotein 2.94 subunit alpha, Mitochondrial precursor (ETFA) Chloride intracellular channel 3.04 protein 1 (CLIC1)

–

P13804

35.058/∼ 30

1.98 [10]

O00299

26.775/∼ 29

5.09/∼ 5.5

134

146

Calcium/phospholipid-binding protein from lipid and vesicular trafficking, anticoagulant protein, inhibitor of the thromboplastin-specific complex. Cell growth inhibitor when transfected in hepatocellular carcinoma cells.

Calcium/phospholipid-binding protein, anticoagulant protein, inhibitor of the phospholipase A2. 5.85/∼ 6.15 Calcium/phospholipid-binding protein, promoting membrane fusion and involved in exocytosis. 8.62/∼ 7.6 Electron acceptor for several dehydrogenases.

179 199

Tumor protein D52 (TPD52) Oncogene DJ1 (PARK7)

3.64 2.35

– 1.34 [1]

P55327 Q99497

19.851/∼ 27 19.878/∼ 24

4.94/∼ 5.2 6.33/∼ 6.6

201

Acyl-protein thioesterase 1 (LYPA1)

2.87

–

O75608

24.653/∼ 24

6.29/∼ 6.9

205

Abhydrolase domaincontaining protein 14B (AB14B) Superoxide dismutase (SODC)

2.44

–

Q96IU4

22.332/∼ 23

5.94/∼ 6.4

1.54

–

P00441

15.795/∼ 18

5.7/∼ 6.1

228

Function

Chloride ion channel protein. Such channels regulate ion traffic between nucleus and cytoplasm and cell cycle (ion homeostasis, transepithelial transport, regulation of electrical excitability, and cell volume). Signaling molecule. RNA binding protein, positive regulator of androgen receptor-dependent transcription, redox-sensitive chaperone, sensor for oxidative stress, with cell-growth promoting and transforming activity. Hydrolase of fatty acids from S-acylated cysteine residues in proteins such as trimeric G alpha proteins or HRAS. Hydrolase.

Cu–Zn binding oxidoreductase, destroying radicals which are normally produced within the cells and which are toxic to biological systems.

D.T. Petrova et al. / Clinical Biochemistry 41 (2008) 1224–1236

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Table 2 (continued ) Spot label

Protein name

Acc. no.

Mr, kDa Calc./Obs.

pI Calc./Obs.

Function

Present study

Previous studies

240

Peroxiredoxin-5, mitochondrial precursor (PRDX5)

2.01

4.39 [10]

P30044

22.012/∼ 17

8.85/∼7.9

3.74

–

Q01469

15.155/∼ 14

6.84/∼6.8

Fatty acid-binding protein, liver (FABPL)

4.07

2.5 [8]; 0.39 [9]

P07148

14.199/∼ 9

6.6/∼ 6.1

255

Fatty acid-binding protein, liver (FABPL)

3.08

2.5 [8]; 0.39 [9]

P07148

14.199/∼ 9

6.6/∼ 6.6

60 L

Vimentin (VIME)

0.42

53.488/∼ 46

5.06/∼5

60 R

Desmin (DESM)

0.48

4.78 [6]; 0.51 [1]; P08670 0.25 [8]; negative in cancer [11]; 7.54, 0.38 [10] 0.23, 0.3 [1]; P17661 negative in polyp mucosa, 0.34 [8]

Oxidoreductase, reducing hydrogen peroxide and alkyl hydroperoxides with reducing equivalents provided through the thioredoxin system; involved in apoptosis. Protein with highest affinity for C18 chain length, decreases the chain length or introduces double bonds. May be involved in keratinocyte differentiation. This protein binds free fatty acids and their coenzyme A derivatives, bilirubin, and some other small molecules in the cytoplasm. May be involved in intracellular lipid transport. This protein binds free fatty acids and their coenzyme A derivatives, bilirubin, and some other small molecules in the cytoplasm. May be involved in intracellular lipid transport. Intermediate filament, found in various non-epithelial cells, especially mesenchymal cells.

244

Fatty acid-binding protein, epidermal (FABPE)

254

53.503/∼ 45

5.21/∼5.2

94 L

Tropomyosin beta chain (TPM2)

0.21

0.62 [10]

P07951

32.831/∼ 40

94 RO Tropomyosin alpha-3 chain (TPM3)

0.31

–

P06753

32.799/∼ 40

94 RU Tropomyosin-1 alpha chain (TPM1)

0.31

3.44 [1]; 3.99 [10]

P09493

32.689/∼ 39

202

Transgelin (TAGL)

0.28

4.41 [7]; 0.69, 0.89, Q01995 1.06 [1]

22.465/∼ 23

215

Transgelin (TAGL)

0.40

4.41 [7]; 0.69, 0.89, Q01995 1.06 [1]

22.465/∼ 22

219

Transgelin (TAGL)

0.21

4.41 [7]; 0.69, 0.89, Q01995 1.06 [1]

22.465/∼ 20

222

Transgelin (TAGL)

0.21

4.41 [7]; 0.69, 0.89, Q01995 1.06 [1]

22.465/∼ 20

223

Transgelin (TAGL)

0.41

4.41 [7]; 0.69, 0.89, Q01995 1.06 [1]

22.465/∼ 20

224

Myosin regulatory light chain 2, smooth muscle isoform (MLRN) Cofilin-2 (COF2)

0.26

–

P24844

19.814/∼ 20

0.58

–

Q9Y281

18.725/∼ 18

229

Average T/N

Intermediate filament, found in muscle cells. In adult striated muscle they form a fibrous network connecting myofibrils to each other. 4.66/∼5 TPM are actin-binding proteins in muscle and non-muscle cells. In association with the troponin complex they play a central 4.68/∼5.1 role in the calcium dependent regulation of striated muscle contraction and in association with caldesmon in smooth muscle 4.69/∼5.05 contraction. In non-muscle cells TPM are implicated in stabilizing cytoskeleton actin filaments. 8.87/∼8.7 Actin cross-linking/gelling protein, involved in calcium interactions and cell contraction. May be involved in replicative senescence. 8.87/∼8.7 Actin cross-linking/gelling protein, involved in calcium interactions and cell contraction. May be involved in replicative senescence. 8.87/∼7.5 Actin cross-linking/gelling protein, involved in calcium interactions and cell contraction. May be involved in replicative senescence. 8.87/∼7.2 Actin cross-linking/gelling protein, involved in calcium interactions and cell contraction. May be involved in replicative senescence. 8.87/∼6.4 Actin cross-linking/gelling protein, involved in calcium interactions and cell contraction. May be involved in replicative senescence. 4.8/∼ 5.2 Regulator of both smooth muscle and nonmuscle-cell contraction. 7.66/∼ 8.1

Controlling protein of reversible actin polymerization and depolymerization in a pH-sensitive manner. (continued on next page)

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Table 2 (continued ) Spot label

Protein name

Average T/N Present study

Previous studies

231

Transgelin (TAGL)

0.31

235

Destrin (DEST)

238 241 245

Acc. no.

Mr, kDa Calc./Obs.

pI Calc./Obs.

Function

4.41 [7]; 0.69, 0.89, 1.06 [1]

Q01995

22.596/∼ 18

8.87/∼ 6.9

0.48

–

P60981

18.362/∼ 17

8.06/∼ 8.1

Destrin (DEST)

0.54

–

P60981

18.493/∼ 17

8.06/∼ 8.7

Myosin light polypeptide 6 (MYL6) Profilin-1 (PROF1)

0.58

–

P60660

16.788/∼ 16

4.56/∼ 4.5

0.48

–

P07737

15.045/∼ 10

8.44/∼ 8.1

Actin cross-linking/gelling protein, involved in calcium interactions and cell contraction. May be involved in replicative senescence. Actin-binding/depolymerizing protein, acting in a pH-independent manner. Actin-binding/depolymerizing protein, acting in a pH-independent manner. Regulatory light chain of myosin, not calcium-binding. Actin-binding protein affecting the cytoskeleton. At high concentrations, profilin prevents the polymerization of actin, whereas it enhances it at low concentrations.

Acc. No — accession number from UniProtKB/SwissProt data base (http://www.ebi.uniprot.org/uniprot-srv/index.do); Mr, kDa — nominal mass in kilodaltons; pI — isoelectric point; Calc. — calculated value; Obs. — observed value.

adhesion, contraction and the cytoskeleton. We observed a consistent up-regulation of CLIC1 (3.04-fold, P b 0.05) and TPD52 (3.64-fold, P b 0.05) in all tumor samples, which were recently identified as early tumor markers in other cancers. Additionally, TPD52 overexpression was confirmed by Western blotting (2.05-fold, P b 0.05) in all samples. The present study should provide a basis for developing markers with clinical relevance to colorectal cancer.

Acknowledgments We thank Christina Wiese, Christa Scholz and Susanne Goldmann for their expert technical assistance. The study was partially supported by Forschungförderungsprogramm 2005 University Medical Center Göttingen, Germany and Bulgarian consortium for structural genomics and in silico drug design (Grant No. DRI-5-2006).

Fig. 3. Validation of proteins differentially expressed in colorectal cancer. Representative Western blot immunochemical analysis using antibodies specific for (a1) TPD52 or (b1) ANXA5. (a2) Percent expression of TPD52 in colon carcinoma tissue relative to unaffected colon, P b 0.05. (b2) Percent expression of ANXA5 in colon carcinoma tissue relative to unaffected colon carcinoma from the same individual, P N 0.05. Data are stratified according to tumor localization.

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Appendix A. Differentially regulated spots among 6 patients with colorectal cancer (6 tumor and 6 normal tissues) Spot label

Average T/N

T/N in 6 patients with colorectal cancer 1 pT2N0M0

Up-regulated proteins in tumor tissue (average T/N N 1.5) 4* 2.43 2.51 9* 2.41 2.15 36 2.30 1.65 53 L* 2.28 3.11 74* 1.92 1.55 125* 3.95 4.24 131* 2.42 2.02 132* 1.90 1.22 134* 1.61 1.44 136 O* 2.12 1.74 136 U* 1.78 1.65 140* 2.94 2.17 146* 3.04 1.61 179* 3.64 1.78 199* 2.35 1.50 201* 2.87 1.92 205* 2.44 2.66 228* 1.54 1.39 240* 2.01 1.54 244* 3.74 2.64 254* 4.07 1.82 255* 3.08 1.50 Down-regulated proteins in tumor tissue (average T/N b 0.6) 60 L* 0.42 0.45 60 R* 0.48 0.67 94 L* 0.21 0.09 94 RO* 0.31 0.28 94 RU* 0.31 0.15 202* 0.28 0.33 215* 0.40 0.69 219* 0.21 0.08 222* 0.21 0.15 223* 0.41 0.25 224* 0.26 0.29 229* 0.58 0.66 231* 0.31 0.22 235* 0.48 0.65 238* 0.54 0.58 241* 0.58 0.59 245* 0.48 0.40

2 pT2N0M0

3 pT2N0M0

4 pT3N0M0

5 pT3N1M1

6 pT4N1M1

3.73 2.16 0.09 1.66 1.58 10.88 2.61 2.29 2.29 1.88 1.56 4.37 2.66 9.98 2.19 5.58 3.25 1.45 3.07 5.05 2.43 1.16

1.81 2.44 2.05 2.58 3.18 3.07 2.59 1.66 1.60 2.69 2.07 3.55 3.01 1.62 1.57 2.47 2.25 1.74 2.08 8.49 3.07 6.95

3.17 1.87 1.87 1.66 2.16 3.04 1.80 1.50 1.52 2.60 1.93 1.98 3.99 2.79 2.09 3.99 2.41 1.47 2.45 2.83 4.81 2.48

2.44 3.09 4.51 2.39 1.75 1.61 2.29 1.83 2.03 2.95 1.91 2.09 2.25 2.18 4.62 2.24 2.12 1.53 1.56 0.87 1.64 1.40

0.92 2.76 3.63 2.31 1.33 0.84 3.23 2.93 0.81 0.89 1.59 3.49 4.70 3.49 2.11 1.04 1.97 1.65 1.36 2.55 10.67 4.99

0.44 0.55 0.09 0.40 0.31 0.13 0.43 0.04 0.23 0.34 0.13 0.63 0.25 0.57 0.58 0.56 0.42

0.42 0.53 0.56 0.56 0.70 0.44 0.42 0.62 0.63 0.52 0.48 0.79 0.47 0.59 0.63 0.64 0.67

0.27 0.17 0.12 0.14 0.23 0.25 0.25 0.08 0.08 0.33 0.12 0.59 0.13 0.48 0.41 0.61 0.60

0.72 0.57 0.16 0.23 0.25 0.26 0.20 0.32 0.14 0.51 0.28 0.55 0.30 0.28 0.56 0.38 0.37

0.25 0.41 0.22 0.26 0.20 0.28 0.44 0.12 0.07 0.53 0.26 0.30 0.50 0.30 0.49 0.68 0.43

T — tumor tissue; N — normal tissue; Average T/N — average ratio of average relative intensity of each spot in 6 patients; T/N — ratio of average relative intensity of each spot; * — P b 0.05 regulated protein expression between tumor and normal tissue using Wilcoxon text.

Appendix B. Score and matched peptides of the identified proteins using nano-LC MS/MS Spot label

Protein ID

Score

No. of matched peptides

Sequence of matched peptides

4

GLU2B

121

6

9

CADH1

99

3

36

KPYM

163

7

LIELQAGKK NKFEEAER SLEDQVEMLR + oxidation (M) ESLQQMAEVTR + oxidation (M) TVKEEAEKPER AQQEQELAADAFK VTEPLDR NTGVISVVTTGLDR GQVPENEANVVITTLK GDYPLEAVR GDLGIEIPAEK SVETLKEMIK + oxidation (M) (continued on next page)

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D.T. Petrova et al. / Clinical Biochemistry 41 (2008) 1224–1236

Appendix B (continued ) Spot label

Protein ID

Score

No. of matched peptides

53 L

EF1G

50

3

74

EFTU

347

12

125

IPYR

61

4

131

VDAC1

85

2

132

ANXA5

486

12

134

GLOD4

88

3

136 O

ANXA3

147

9

136 U

ANXA4

144

12

140

ETFA

346

10

Sequence of matched peptides LDIDSPPITAR ITLDNAYMEK + oxidation (M) NTGIICTIGPASR + carbamidomethyl (C) IYVDDGLISLQVK QVLEPSFR ILGLLDAYLK KLDPGSEETQTLVR IILPPEK EHLLLAR GTVVTGTLER VEAQVYILSK AEAGDNLGALVR TVVTGIEMFHK + oxidation (M) YEEIDNAPEER KGDECELLGHSK + carbamidomethyl (C) LLDAVDTYIPVPAR SLERAEAGDNLGALVR GITINAAHVEYSTAAR TIGTGLVTNTLAMTEEEK + oxidation (M) AAPFSLEYR YVANLFPYK DKDFAIDIIK VIAINVDDPDAANYNDINDVKR LTLSALLDGK VTQSNFAVGYK FITIFGTR VLTEIIASR LYDAYELK GAGTDDHTLIR MLVVLLQANR + oxidation (M) SEIDLFNIRK NFATSLYSMIK + oxidation (M) GTVTDFPGFDER SNAQRQEISAAFK ETSGNLEQLLLAVVK GLGTDEESILTLLTSR SIPAYLAETLYYAMK + oxidation (M) VTLAVSDLQK SLPQSDPVLK ILTPLVSLDTPGK ALLTLADGR LTFDEYR QDAQ ILYK NTPAFLAER MLISILTER + oxidation (M) GIGTDEFTLNR SDTSGDYEITLLK SLGDDISSETSGDFRK DYPDFSPSVDAEAIQK VLVSLSAGGR FLTVLCSR + carbamidomethyl (C) AEIDMLDIR AEIDMLDIR + oxidation (M) GLGTDDNTLIR ISQKDIEQSIK QDAQDLYEAGEK ISQTYQQQYGR DEGNYLDDALVR QDAQDLYEAGEKK AASGFNAMEDAQTLR + oxidation (M) GAGTDEGCLIEILASR + carbamidomethyl (C) SPDTFVR SDRPELTGAK VLVAQHDVYK

D.T. Petrova et al. / Clinical Biochemistry 41 (2008) 1224–1236

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Appendix B (continued ) Spot label

Protein ID

Score

No. of matched peptides

146

CLIC1

277

6

179

TPD52

112

3

199

PARK7

69

3

201

LYPA1

44

3

205

AB14B

162

4/5

228

SODC

150

4

240

PRDX5

100

3

244

FABPE

76

4

254

FABPL

96

3

255

FABPL

71

3

60 L

VIME

27

2

60 R

DESM

216

12

94 L

TPM2

255

10

Sequence of matched peptides VVPEMTEILK + oxidation (M) LGGEVSCLVAGTK + carbamidomethyl (C) TIYAGNALCTVK + carbamidomethyl (C) LEVAPISDIIAIK VAAKLEVAPISDIIAIK LLYDLADQLHAAVGASR AAVDAGFVPNDMQVGQTGK + oxidation (M) YLSNAYAR GFTIPEAFR IGNCPFSQR + carbamidomethyl (C) GVTFNVTTVDTK NSNPALNDNLEK LAALNPESNTAGLDIFAK TSETLSQAGQK SFEEKVENLK ASAAFSSVGSVITK DGLILTSR DVVICPDASLEDAKK + carbamidomethyl (C) GAEEMETVIPVDVMR + 2 oxidation (M) ALIDQEVK TLVNPANVTFK ASFPQGPIGGANR EALPGSGQAR INAANYASVK AVAIDLPGLGHSK AVAIDLPGLGHSK EGTIQVQGQALFFR HVGDLGNVTADK ADDLGKGGNEESTK TLVVHEKADDLGK GDGPVQGIINFEQK LLADPTGAFGK FSMVVQDGIVK FSMVVQDGIVK + oxidation (M) ELGVGIALR FEETTADGR TTQFSCTLGEK + carbamidomethyl (C) LVVECVMNNVTCTR + 2 carbamidomethyl (C) FTITAGSK TVVQLEGDNK AIGLPEELIQK FTITAGSK TVVQLEGDNK AIGLPEELIQK QQYESVAAK QDVDNASLAR LLEGEESR AQYETIAAK ADVDAATLAR VSDLTQAANK QVEVLTNQR DNLLDDLQR EYQDLLNVK VAELYEEELR FASEASGYQDNIAR FLEQQNAALAAEVNR VDVERDNLLDDLQR DGEVVSEATQQQHEVL LDKENAIDR LVILEGELER IQLVEEELDR KLVILEGELER ATDAEADVASLNR QLEEEQQALQK (continued on next page)

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D.T. Petrova et al. / Clinical Biochemistry 41 (2008) 1224–1236

Appendix B (continued ) Spot label

Protein ID

Score

No. of matched peptides

94 RO

TPM3

262

5/8

94 RU

TPM1

342

10/14

202

TAGL

274

7

215

TAGL

102

7

219

TAGL

289

7

222

TAGL

370

9

223

TAGL

171

4

224

MLRN

185

7

Sequence of matched peptides LATALQKLEEAEK LEEAEKAADESER ATDAEADVASLNRR IQLVEEELDRAQER IQLVEEELDR LATALQKLEEAEK LATALQKLEEAEK LEEAEKAADESER LEEAEKAADESER LVIIEGDLERTEER TQLEEELDRAQER TQLEEELDRAQER CAELEEELK + carbamidomethyl (C) LVIIESDLER AEERAELSEGK AEERAELSEGK IQLVEEELDR KLVIIESDLER LATALQKLEEAEK LATALQKLEEAEK LEEAEKAADESER LEEAEKAADESER LVIIESDLERAEER IQLVEEELDRAQER IQLVEEELDRAQER CAELEEELKTVTNNLK + carbamidomethyl (C) AAEDYGVIK TLMALGSLAVTK KYDEELEER TLMALGSLAVTK + oxidation (M) QMEQVAQFLK + oxidation (M) EFTESQLQEGK TDMFQTVDLFEGK + oxidation (M) AAEDYGVIK YDEELEER KYDEELEER TLMALGSLAVTK + oxidation (M) QMEQVAQFLK + oxidation (M) VPENPPSMVFK + oxidation (M) EFTESQLQEGK AAEDYGVIK YDEELEER KYDEELEER TLMALGSLAVTK + oxidation (M) QMEQVAQFLK + oxidation (M) EFTESQLQEGK TDMFQTVDLFEGK + oxidation (M) AAEDYGVIK YDEELEER KYDEELEER TLMALGSLAVTK + oxidation (M) QMEQVAQFLK + oxidation (M) VPENPPSMVFK + oxidation (M) EFTESQLQEGK LVNSLYRDGSKPVK TDMFQTVDLFEGK + oxidation (M) YDEELEER KYDEELEER TLMALGSLAVTK + oxidation (M) EFTESQLQEGK ELLTTMGDR + oxidation (M) LNGTDPEDVIR GNFNYVETR EAFNMIDQNR + oxidation (M)

D.T. Petrova et al. / Clinical Biochemistry 41 (2008) 1224–1236

1235

Appendix B (continued ) Spot label

Protein ID

Score

No. of matched peptides

229

COF2

97

4

231

TAGL

133

6

235 238

DEST DEST

47 61

1 3

241

MYL6

207

5/6

245

PROF1

153

6

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Sequence of matched peptides FTDEEVDEMYR + oxidation (M) ATSNVFAMFDQSQIQEFK + oxidation (M) ELLTTMGDRFTDEEVDEMYR + 2 oxidation (M) LLPLNDCR + Carbamidomethyl carbamidomethyl (C) YALYDATYETK LGGNVVVSLEGKPL QILVGDIGDTVEDPYTSFVK YDEELEER KYDEELEER TLMALGSLAVTK + oxidation (M) QMEQVAQFLK + oxidation (M) VPENPPSMVFK + oxidation (M) TDMFQTVDLFEGK + oxidation (M) YALYDASFETK IFYDMK + oxidation (M) CIIVEEGK + carbamidomethyl (C) YALYDASFETK HVLVTLGEK EAFQLFDR ILYSQCGDVMR + carbamidomethyl (C); oxidation (M) ILYSQCGDVMR + carbamidomethyl (C); oxidation (M) DQGTYEDYVEGLR NKDQGTYEDYVEGLR TLVLLMGK + oxidation (M) DSPSVWAAVPGK STGGAPTFNVTVTK DSLLQDGEFSMDLR + oxidation (M) TFVNITPAEVGVLVGK SSFYVNGLTLGGQK

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