**2.6.6 2DE in identification of bladder carcinoma protein marker (Calreticulin)**

Susumu Kageyama et al., 2004 screened proteins as tumor markers for bladder cancer by proteomic analysis (2DE) of cancerous and healthy tissues and investigated the diagnostic accuracy of one such marker, *Calreticulin* (CRT) in urine.

They have produced two important findings in their experiments. The first is that increased production of CRT in bladder cancer tissue which was confirmed by proteome profiling by 2DE. Furthermore, we detected two isoforms of CRT, and full-length CRT which was more useful than cleaved CRT for distinguishing bladder cancer from healthy tissue.

In their study, although visual comparison of 2DE gels of TCC (transitional cell carcinoma) and noncancerous urothelium showed similar expression profiles, 15 protein spots (U-1 to U-15) were more intense in TCC samples (Fig. 1). They identified 10 of the proteins by use of a peptide mass fingerprinting method. One spot among them, with an apparent mass of 55 kDa and pI of 4.3, was identified as CRT (spot U-2 in Fig. 1 A). From NH2-terminal amino acid sequencing, 10 amino acids were sequenced (EPAVYFKEQF), and they were identical to residues 1–10 of mature human CRT according to the sequence homology search.

Further, to validate the 2DE finding of increased production of full-length CRT in bladder cancer tissue, they performed quantitative Western blot analysis in cancerous and healthy tissue using anti-COOH-terminus antibody. They compared CRT band intensities for 22 cancerous with 10 noncancerous tissues. For band quantification, they defined the CRT band derived from a total of 1 µg of heat-shocked HeLa cell extract as 1.0 unit/µg of protein. The mean (SD) concentrations in cancerous and healthy tissue were 1.0 (0.4) and 0.4 (0.3) units/µg of protein, respectively (Mann–Whitney *U*-test, *P* = 0.0003; Fig. 2). Among these tissue samples, six pairs of cancerous and noncancerous specimens were obtained from the bladders of patients who had undergone radical cystectomy. CRT concentrations were higher in all cancer tissues compared with the corresponding healthy urothelium.

from spot cutting to protein digestion to MS analysis and image annotation is reduced by over 50% compared to manual processing of gel samples, with a corresponding reduction in error. All of the instrumentation and software in this process is part of the integrated

MALDI-TOF MS provides an ideal high-throughput solution for protein identification; however, where protein identity is ambiguous, known databases must be searched with a higher degree of sequence information. The Micromass Q-Tof family of MS-MS instruments incorporates quadrupole/ orthogonal acceleration time-of-flight (Q/oa-TOF) technology, enabling exact mass measurement, and acquisition of the highest-level peptide sequence information for de novo sequencing and BLAST analyses. Protein digest samples in microtiter plates, prepared with the Mass*PREP* station, can be transferred directly to the Micromass CapLC (capillary HPLC) system for automated injection into the Q-Tof *micro* for integrated LC-MS-MS under MassLynx software control. The capability for MS to MS-MS switching "on the fly" with the Q-Tof family of instruments maximizes the amount of amino acid sequence information that can be generated with these instruments. MassSeq software also provides the

ProteomeWorks system, a set of powerful tools for proteomic analysis.

**2.6.5 Advanced protein characterization with ESI-LC-MS and MALDI-TOF** 

capacity for automated de novo amino acid sequencing based on the MS results.

useful than cleaved CRT for distinguishing bladder cancer from healthy tissue.

to residues 1–10 of mature human CRT according to the sequence homology search.

higher in all cancer tissues compared with the corresponding healthy urothelium.

accuracy of one such marker, *Calreticulin* (CRT) in urine.

**2.6.6 2DE in identification of bladder carcinoma protein marker (Calreticulin)** 

Susumu Kageyama et al., 2004 screened proteins as tumor markers for bladder cancer by proteomic analysis (2DE) of cancerous and healthy tissues and investigated the diagnostic

They have produced two important findings in their experiments. The first is that increased production of CRT in bladder cancer tissue which was confirmed by proteome profiling by 2DE. Furthermore, we detected two isoforms of CRT, and full-length CRT which was more

In their study, although visual comparison of 2DE gels of TCC (transitional cell carcinoma) and noncancerous urothelium showed similar expression profiles, 15 protein spots (U-1 to U-15) were more intense in TCC samples (Fig. 1). They identified 10 of the proteins by use of a peptide mass fingerprinting method. One spot among them, with an apparent mass of 55 kDa and pI of 4.3, was identified as CRT (spot U-2 in Fig. 1 A). From NH2-terminal amino acid sequencing, 10 amino acids were sequenced (EPAVYFKEQF), and they were identical

Further, to validate the 2DE finding of increased production of full-length CRT in bladder cancer tissue, they performed quantitative Western blot analysis in cancerous and healthy tissue using anti-COOH-terminus antibody. They compared CRT band intensities for 22 cancerous with 10 noncancerous tissues. For band quantification, they defined the CRT band derived from a total of 1 µg of heat-shocked HeLa cell extract as 1.0 unit/µg of protein. The mean (SD) concentrations in cancerous and healthy tissue were 1.0 (0.4) and 0.4 (0.3) units/µg of protein, respectively (Mann–Whitney *U*-test, *P* = 0.0003; Fig. 2). Among these tissue samples, six pairs of cancerous and noncancerous specimens were obtained from the bladders of patients who had undergone radical cystectomy. CRT concentrations were To confirm the presence of isoforms of CRT, they performed two-dimensional Western blotting with two different antibodies: monoclonal antibody FMC75, which was produced against recombinant human CRT; and a polyclonal antibody that was produced using synthesized peptides of the human CRT COOH terminus (amino acids 388–400) as an immunogen. On Western blots with anti- COOH-terminus antibody, only one of the two spots was visualized, whereas both spots became visible on blots incubated with FMC75 (Fig. 3). One was the same as the 55-kDa (pI 4.3) spot, and the other had an apparent molecular mass of 40 kDa and pI of 4.5. This lower molecular- mass spot had the same NH2 terminal amino acid sequence as amino acids 1–10 of mature human CRT as shown by amino acid sequencing. Therefore, they suggested that the higher-molecular-mass spot was the full-length form and the other spot was a cleaved form that is truncated elsewhere in the COOH domain. Production of the full-length CRT in cancer tissue was increased compared with in healthy tissue, but the spots for cleaved CRT in cancerous and healthy urothelium had intensities that were similar and were reproducible on all silverstained 2DE gels.

Subsequently they tried to confirm whether anti-COOH terminus antibody binds to molecules other than full lengt1h CRT and performed immuno-precipitations (Fig. 4). The Western blot of the immuno-precipitate extracted from cancer tissue revealed only one band, and they concluded that anti-COOH-terminus antibody binds specifically to fulllength CRT of ~55 kDa. They therefore judged that full-length CRT recognized by anti-COOH terminus antibody is appropriate as a tumor marker.

Fig. 1. Silver-stained images of analytical narrow-pH-range 2DE gels of proteins from bladder cancer. (A), pH 4.0–5.0; (B), pH 4.5–5.5; (C), pH 5.0–6.0; (D), pH 5.5–6.7. Arrows indicate spots (U-1 to U-15) containing higher amounts of protein. Spot U-2, ~55 kDa and pI 4.3, was confirmed to be CRT by a peptide mass fingerprinting method and NH2-terminal amino acid sequencing. *(Source: Susumu Kageyama et al., 2004 Clinical Chemistry, 50: 857–866)*

Two Dimensional Gel Electrophoresis in Cancer Proteomics 379

Fig. 4. Western blot with FMC75 antibody. *Arrow* indicates 55-kDa full-length CRT. *Lane 1*, total cell lysate; *lane 2*, extraction from protein A Sepharose beads that did not bind antibody; *lane 3*, immunoprecipitant eluted from beads binding anti-COOH-terminus antibody; *lane 4*, immunoprecipitant extracted from beads binding normal rabbit IgG indicates approximate molecular masses. *(Source: Susumu Kageyama et al., 2004 Clinical* 

Leucine-rich alpha-2-glycoprotein is characterized by its unusually high content of leucine, about 17% by weight. The primary structure of LRG suggests that it may be a membrane associated or membrane-derived protein. Aberrant regulation of LRG has been observed in

Tatsuhiko Kakisaka et al., 2007 examined the plasma LRG expression levels of cancer patients who were not in the acute inflammatory phase. First, they selected cancer patients with normal C-reactive protein (CRP) concentration (Table 1, P6–10) to explore whether the increase in LRG levels paralleled the dynamics of the commonacute phase proteins. Second, they tested plasma from cancer patients with a normal level of CA19-9 a tumor marker commonly used for the diagnosis of pancreatic cancer, to examine the possibility of plasma

SDS-PAGE/Western blotting using an anti-LRG antibody showed consistent upregulation of LRG in these patients, who were negative for CRP and/or CA19-9, compared with the noncancer bearing healthy donors (Fig. 5). Therefore, increased amounts of LRG may be independent of the regulation of other acute phase proteins and tumor markers. They also examined plasma samples from chronic pancreatitis patients and found that they tended to express lower LRG levels compared with the samples from pancreatic cancer patients. By correlating the expression levels of LRG with clinical information from a large sample set, they hope to validate the utility of LRG as a biomarker to monitor the status of patients. Some plasma samples from pancreatic cancer patients did not express high LRG levels, leading us to suggest that the examination of plasma LRG levels in combination with the existing biomarkers would increase the

**2.6.7 2-DE in identification of pancreatic carcinoma protein marker** 

35

LRG levels being using in a way complementary to existing tumor markers.

patients with malignant disease and with virus infection.

specificity and sensitivity of the diagnosis.

*Chemistry, 50: 857–866)*

Fig. 2. Quantitative Western blot analysis of cancerous and healthy tissues using anti-COOH-terminus antibody. *Lines* show six pairs of cancerous and healthy specimens obtained from the bladders of patients who had undergone radical cystectomy.*(Source:* Susumu Kageyama *et al., 2004 Clinical Chemistry, 50: 857–866)*

Fig. 3. Close-up sections of two-dimensional Western blot images obtained with two different antibodies, FMC75 (*A*) and anti-COOH terminus antibody (*B*), and proteins in silver-stained pH 4–7 gels (*C*). *Left panels* are bladder cancer and *right panels* are healthy urothelium. *Arrows* indicate full-length CRT (55 kDa; pI 4.3), and *arrowheads* indicate cleaved CRT (40 kDa; pI 4.5). These two CRT forms have the same NH2-terminal amino acid sequence (EPAVYFKEQF), but cleaved CRT is considered to lack the COOH terminus because of no immunoreactivity for anti-COOH-terminus antibody. *(Source: Susumu Kageyama et al., 2004 Clinical Chemistry, 50: 857–866)*

378 Gel Electrophoresis – Advanced Techniques

Fig. 2. Quantitative Western blot analysis of cancerous and healthy tissues using anti-COOH-terminus antibody. *Lines* show six pairs of cancerous and healthy specimens obtained from the bladders of patients who had undergone radical cystectomy.*(Source:*

Fig. 3. Close-up sections of two-dimensional Western blot images obtained with two different antibodies, FMC75 (*A*) and anti-COOH terminus antibody (*B*), and proteins in silver-stained pH 4–7 gels (*C*). *Left panels* are bladder cancer and *right panels* are healthy urothelium. *Arrows* indicate full-length CRT (55 kDa; pI 4.3), and *arrowheads* indicate cleaved

CRT (40 kDa; pI 4.5). These two CRT forms have the same NH2-terminal amino acid sequence (EPAVYFKEQF), but cleaved CRT is considered to lack the COOH terminus because of no immunoreactivity for anti-COOH-terminus antibody. *(Source: Susumu* 

*Kageyama et al., 2004 Clinical Chemistry, 50: 857–866)*

Susumu Kageyama *et al., 2004 Clinical Chemistry, 50: 857–866)*

Fig. 4. Western blot with FMC75 antibody. *Arrow* indicates 55-kDa full-length CRT. *Lane 1*, total cell lysate; *lane 2*, extraction from protein A Sepharose beads that did not bind antibody; *lane 3*, immunoprecipitant eluted from beads binding anti-COOH-terminus antibody; *lane 4*, immunoprecipitant extracted from beads binding normal rabbit IgG indicates approximate molecular masses. *(Source: Susumu Kageyama et al., 2004 Clinical Chemistry, 50: 857–866)*

### **2.6.7 2-DE in identification of pancreatic carcinoma protein marker**

Leucine-rich alpha-2-glycoprotein is characterized by its unusually high content of leucine, about 17% by weight. The primary structure of LRG suggests that it may be a membrane associated or membrane-derived protein. Aberrant regulation of LRG has been observed in patients with malignant disease and with virus infection.

Tatsuhiko Kakisaka et al., 2007 examined the plasma LRG expression levels of cancer patients who were not in the acute inflammatory phase. First, they selected cancer patients with normal C-reactive protein (CRP) concentration (Table 1, P6–10) to explore whether the increase in LRG levels paralleled the dynamics of the commonacute phase proteins. Second, they tested plasma from cancer patients with a normal level of CA19-9 a tumor marker commonly used for the diagnosis of pancreatic cancer, to examine the possibility of plasma LRG levels being using in a way complementary to existing tumor markers.

SDS-PAGE/Western blotting using an anti-LRG antibody showed consistent upregulation of LRG in these patients, who were negative for CRP and/or CA19-9, compared with the noncancer bearing healthy donors (Fig. 5). Therefore, increased amounts of LRG may be independent of the regulation of other acute phase proteins and tumor markers. They also examined plasma samples from chronic pancreatitis patients and found that they tended to express lower LRG levels compared with the samples from pancreatic cancer patients. By correlating the expression levels of LRG with clinical information from a large sample set, they hope to validate the utility of LRG as a biomarker to monitor the status of patients. Some plasma samples from pancreatic cancer patients did not express high LRG levels, leading us to suggest that the examination of plasma LRG levels in combination with the existing biomarkers would increase the specificity and sensitivity of the diagnosis.

Two Dimensional Gel Electrophoresis in Cancer Proteomics 381

Fig. 5. Elevated level of plasma LRG in pancreatic cancer. The localizations of the five LRG spots are indicated by arrows 1–5 on the 2D image of the 150mM NaCl sample (A). The boxed area was transferred to a nitrocellulose membrane and scanned with a laser scanner to obtain the LRG spots on the membrane (B). The scanned membrane was reacted with an anti-LRG antibody and the antibody–antigen complexes were detected with an ECL system (C). The fluorescent signals of the LRG spots on the 2D-PAGE gels were compared between non-cancer bearing healthy donors (D) and pancreatic cancer patients (E). *(Source: Tatsuhiko* 

**2.6.9 2DE in identification of squamous cervical carcinoma protein markers** 

Recently, proteomic and genomic approaches to identify tumor markers are undergoing. Hellman and coworkers reported the protein expression patterns in primary carcinoma of the vagina. In relation to HPV, C33A cell line transfected with HPV *E7* gene and proteomic and genomic analyses were performed. But, until now, there was no report of SCC in cervix tissues. Prof. W.S. Ahn and his colleagues at Cancer Research Center of The Catholic University of Korea, South Korea contributed much more to understand the significance of 2DGE in diagnosis of cervical cancer. In general, screening in cervical cancer is progressing to find out candidate genes and proteins, which may work as biological markers and play a role in tumor progression. They examined the protein expression patterns of squamous cell carcinoma (SCC) tissues from Korean women using two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of fight (MALDI-TOF) mass spectrometer. A total of 35 proteins are detected in SCC. 17 proteins are up regulated and 18 proteins are down-regulated. Among the proteins identified, 12 proteins (pigment epithelium derived factor, annexin A2 and A5, keratin 19 and 20, heat shock protein 27, smooth muscle protein 22 alpha, alpha-enolase, squamous cell carcinoma antigen 1 and 2, glutathione S-transferase, apolipoprotein a1) are previously known proteins involved in tumor and 21 proteins were newly identified in this study. They concluded that the 2-DE offers total protein expression profiles of SCC tissues and further characterization of proteins that are differentially expressed will give a chance to identify tumor-specific

*Kakisaka et al., 2007 Journal of Chromatography B, 852: 257–267)* 

diagnostic markers for SCC (Fig. 7 & 8).

Some protein spots on 2D-PAGE gels overlapped across fractions in the anion-exchange chromatography even when they used the step-wise gradient method with system wash between intervals. They considered these overlapping spots to correspond to different isoforms of the same protein, and have therefore counted all protein spots on the 2D-PAGE gels. However, not every differentially expressed protein was considered to be a suitable tumor marker; for example, spots 8 and 11 (transthyretin) were differentially expressed between cancer patients and healthy donors, but they were very minute amounts of the total abundant transthyretin, and it was difficult to extract theseportion of the protein. On the contrary, LRG, which was also differentially expressed between cancer patients and healthy donors, was only expressed in one fraction and was therefore selected as a candidate for a tumor marker of pancreatic cancer.

The use of high-resolution 2D-PAGE with narrow-range IPG gels and large-format second dimension gels could solve this problem to some extent.


Table 1. Patient informations of validation set 1

#### **2.6.8 2-DE in identification of human gastric carcinoma protein marker**

Gastric cancer is the second most common cause of cancer deaths worldwide and due to its poor prognosis, it is important that specific biomarkers are identified to enable its early detection. Through 2-D gel electrophoresis and MALDI-TOF-TOF-based proteomics approaches, Chien-Wei Tseng et al., 2011 found that 14-3-3β, which was one of the proteins that were differentially expressed by 5-fluorouracil-treated gastric cancer SC-M1 cells, was up regulated in gastric cancer cells. 14-3-3β levels in tissues and serum were further validated in gastric cancer patients and controls. The results showed that 14-3-3β levels were elevated in tumor tissues in comparison to normal tissues, and serum levels in cancer patients were also significantly higher than those in controls (Fig. 6). Elevated serum 14-3-3β levels highly correlated with the number of lymph node metastases, tumor size, and a reduced survival rate. Moreover, over-expression of 14-3-3β enhanced the growth, invasiveness, and migratory activities of tumor cells. Twenty-eight proteins involved in antiapoptosis and tumor progression were also found to be differentially expressed in 14-3-3βoverexpressing gastric cancer cells. Overall, these results highlight the significance of 14-3- 3β in gastric cancer cell progression and suggest that it has the potential to be used as a diagnostic and prognostic biomarker in gastric cancer.

Some protein spots on 2D-PAGE gels overlapped across fractions in the anion-exchange chromatography even when they used the step-wise gradient method with system wash between intervals. They considered these overlapping spots to correspond to different isoforms of the same protein, and have therefore counted all protein spots on the 2D-PAGE gels. However, not every differentially expressed protein was considered to be a suitable tumor marker; for example, spots 8 and 11 (transthyretin) were differentially expressed between cancer patients and healthy donors, but they were very minute amounts of the total abundant transthyretin, and it was difficult to extract theseportion of the protein. On the contrary, LRG, which was also differentially expressed between cancer patients and healthy donors, was only expressed in one fraction and was therefore selected as a candidate for a

The use of high-resolution 2D-PAGE with narrow-range IPG gels and large-format second

tumor marker of pancreatic cancer.

dimension gels could solve this problem to some extent.

Table 1. Patient informations of validation set 1

diagnostic and prognostic biomarker in gastric cancer.

**2.6.8 2-DE in identification of human gastric carcinoma protein marker** 

Gastric cancer is the second most common cause of cancer deaths worldwide and due to its poor prognosis, it is important that specific biomarkers are identified to enable its early detection. Through 2-D gel electrophoresis and MALDI-TOF-TOF-based proteomics approaches, Chien-Wei Tseng et al., 2011 found that 14-3-3β, which was one of the proteins that were differentially expressed by 5-fluorouracil-treated gastric cancer SC-M1 cells, was up regulated in gastric cancer cells. 14-3-3β levels in tissues and serum were further validated in gastric cancer patients and controls. The results showed that 14-3-3β levels were elevated in tumor tissues in comparison to normal tissues, and serum levels in cancer patients were also significantly higher than those in controls (Fig. 6). Elevated serum 14-3-3β levels highly correlated with the number of lymph node metastases, tumor size, and a reduced survival rate. Moreover, over-expression of 14-3-3β enhanced the growth, invasiveness, and migratory activities of tumor cells. Twenty-eight proteins involved in antiapoptosis and tumor progression were also found to be differentially expressed in 14-3-3βoverexpressing gastric cancer cells. Overall, these results highlight the significance of 14-3- 3β in gastric cancer cell progression and suggest that it has the potential to be used as a

Fig. 5. Elevated level of plasma LRG in pancreatic cancer. The localizations of the five LRG spots are indicated by arrows 1–5 on the 2D image of the 150mM NaCl sample (A). The boxed area was transferred to a nitrocellulose membrane and scanned with a laser scanner to obtain the LRG spots on the membrane (B). The scanned membrane was reacted with an anti-LRG antibody and the antibody–antigen complexes were detected with an ECL system (C). The fluorescent signals of the LRG spots on the 2D-PAGE gels were compared between non-cancer bearing healthy donors (D) and pancreatic cancer patients (E). *(Source: Tatsuhiko Kakisaka et al., 2007 Journal of Chromatography B, 852: 257–267)* 

#### **2.6.9 2DE in identification of squamous cervical carcinoma protein markers**

Recently, proteomic and genomic approaches to identify tumor markers are undergoing. Hellman and coworkers reported the protein expression patterns in primary carcinoma of the vagina. In relation to HPV, C33A cell line transfected with HPV *E7* gene and proteomic and genomic analyses were performed. But, until now, there was no report of SCC in cervix tissues. Prof. W.S. Ahn and his colleagues at Cancer Research Center of The Catholic University of Korea, South Korea contributed much more to understand the significance of 2DGE in diagnosis of cervical cancer. In general, screening in cervical cancer is progressing to find out candidate genes and proteins, which may work as biological markers and play a role in tumor progression. They examined the protein expression patterns of squamous cell carcinoma (SCC) tissues from Korean women using two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of fight (MALDI-TOF) mass spectrometer. A total of 35 proteins are detected in SCC. 17 proteins are up regulated and 18 proteins are down-regulated. Among the proteins identified, 12 proteins (pigment epithelium derived factor, annexin A2 and A5, keratin 19 and 20, heat shock protein 27, smooth muscle protein 22 alpha, alpha-enolase, squamous cell carcinoma antigen 1 and 2, glutathione S-transferase, apolipoprotein a1) are previously known proteins involved in tumor and 21 proteins were newly identified in this study. They concluded that the 2-DE offers total protein expression profiles of SCC tissues and further characterization of proteins that are differentially expressed will give a chance to identify tumor-specific diagnostic markers for SCC (Fig. 7 & 8).

Two Dimensional Gel Electrophoresis in Cancer Proteomics 383

Fig. 7. Comparison of proteome by two-dimensional gel electrophoresis on normal tissues and cervical SCC tissues. Representative examples of 2-DE gels derived from a normal cervix tissue and cervical SCC tissue. Normal cervix (A) and cervical SCC (B) total proteins were separated by 2-DE using IPG strips pH 3–10 in the first and 12% SDS-PAGE in the second dimension. Identified protein spots are indicated by numbers. Proteins down- (A) or up-regulated (B) in cervical SCC are indicated. (*Source: Bae et al., 2005 Gynecologic Oncol., 99:* 

Franzen *et al.,* 1997 well documented that the two-dimensional electrophoresis (2-DE) analyses of human breast carcinoma reveals the following observations: (i) Analysis of samples from different areas of the same tumor showed a high degree of similarity in the pattern of polypeptide expression. Similarly, analysis of two tumors and their metastases revealed similar 2-DE profiles. (ii) In contrast, large variations have been observed between different lesions with comparable histological characteristics. Larger differences in polypeptide expression are pointed out in between potentially highly malignant carcinomas and comparisons of less malignant lesions. These differences are in the same order of magnitude as those observed comparing a breast carcinoma to a lung carcinoma. (iii) The levels of all cytokeratin forms resolved (CK7, CK8, CK15, and CK18) were significantly lower in carcinomas compared to fibro adenomas. (iv) The levels of high molecular weight tropo-myosins (1–3) were lower in carcinomas compared to fibro adenomas. The expression of tropomyosin-1 is 1.7-fold higher in primary tumors with metastatic spread to axillar lymph nodes compared to primary tumors with no evidence of metastasis (*p* < 0.05). (v) The expression of proliferating cell nuclear antigen (PCNA) and some members of the stress protein family (pHSP60, HSP90, and calreticulin) are higher in carcinomas. We conclude that malignant progression of breast carcinomas results in large heterogeneity in polypeptide expression between different tumors, but that some common themes such as

*26-35)* 

**2.6.10 Breast cancer protein profiling** 

Fig. 6. 14-3-3β is differentially expressed after 5-FU treatment of SC-M1 cells. (A) Proteins from the 5-FU-treated (right) SC-M1 cells and the untreated control (left) were compared using 2-DE. Enlarged images and 3-D profiles of 14-3-3b on the gels are shown in (B) and (C). (D) 14-3-3β expression was significantly reduced after 5-FU treatment as confirmed by Western blot. *(Source: Chien-Wei Tseng et al., 2011 Proteomics, 11:2423–2439)* 

In addition to cervical cancer, the 2-dimensional polyacrylamide gel electrophoresis (2-DE) has also been used to examine heterogeneity of protein expression in tissues from different tumors such as bladder, breast, colon–rectum, lung, and ovary. The advantage of 2-DE is that the complex protein expression is analyzed qualitatively and quantitatively. 2-DE combined with MALDI-TOF-MS has been applied to identify cancer-specific protein markers. These markers can provide a basis for developing new methods for early diagnosis and treatment.

Fig. 6. 14-3-3β is differentially expressed after 5-FU treatment of SC-M1 cells.

*11:2423–2439)* 

and treatment.

(A) Proteins from the 5-FU-treated (right) SC-M1 cells and the untreated control (left) were compared using 2-DE. Enlarged images and 3-D profiles of 14-3-3b on the gels are shown in (B) and (C). (D) 14-3-3β expression was significantly reduced after 5-FU treatment as confirmed by Western blot. *(Source: Chien-Wei Tseng et al., 2011 Proteomics,* 

In addition to cervical cancer, the 2-dimensional polyacrylamide gel electrophoresis (2-DE) has also been used to examine heterogeneity of protein expression in tissues from different tumors such as bladder, breast, colon–rectum, lung, and ovary. The advantage of 2-DE is that the complex protein expression is analyzed qualitatively and quantitatively. 2-DE combined with MALDI-TOF-MS has been applied to identify cancer-specific protein markers. These markers can provide a basis for developing new methods for early diagnosis

Fig. 7. Comparison of proteome by two-dimensional gel electrophoresis on normal tissues and cervical SCC tissues. Representative examples of 2-DE gels derived from a normal cervix tissue and cervical SCC tissue. Normal cervix (A) and cervical SCC (B) total proteins were separated by 2-DE using IPG strips pH 3–10 in the first and 12% SDS-PAGE in the second dimension. Identified protein spots are indicated by numbers. Proteins down- (A) or up-regulated (B) in cervical SCC are indicated. (*Source: Bae et al., 2005 Gynecologic Oncol., 99: 26-35)* 

#### **2.6.10 Breast cancer protein profiling**

Franzen *et al.,* 1997 well documented that the two-dimensional electrophoresis (2-DE) analyses of human breast carcinoma reveals the following observations: (i) Analysis of samples from different areas of the same tumor showed a high degree of similarity in the pattern of polypeptide expression. Similarly, analysis of two tumors and their metastases revealed similar 2-DE profiles. (ii) In contrast, large variations have been observed between different lesions with comparable histological characteristics. Larger differences in polypeptide expression are pointed out in between potentially highly malignant carcinomas and comparisons of less malignant lesions. These differences are in the same order of magnitude as those observed comparing a breast carcinoma to a lung carcinoma. (iii) The levels of all cytokeratin forms resolved (CK7, CK8, CK15, and CK18) were significantly lower in carcinomas compared to fibro adenomas. (iv) The levels of high molecular weight tropo-myosins (1–3) were lower in carcinomas compared to fibro adenomas. The expression of tropomyosin-1 is 1.7-fold higher in primary tumors with metastatic spread to axillar lymph nodes compared to primary tumors with no evidence of metastasis (*p* < 0.05). (v) The expression of proliferating cell nuclear antigen (PCNA) and some members of the stress protein family (pHSP60, HSP90, and calreticulin) are higher in carcinomas. We conclude that malignant progression of breast carcinomas results in large heterogeneity in polypeptide expression between different tumors, but that some common themes such as

Two Dimensional Gel Electrophoresis in Cancer Proteomics 385

Fig. 9. Sensitivity of 2D-DIGE and compatibility with SYPRO gel staining. *A*, comparison of 2D-DIGE imaging and SyproRuby poststaining. Merged Cy dye image of HB4a lysate

Fig. 10. 2D-DIGE is a reproducible detection method. Duplicate samples of HB4a and

High-resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a powerful research tool for the analytical separation of cellular proteins. The qualitative and quantitative pattern of polypeptides synthesized by a cell represents its phenotype and thus defines characteristics such as the morphology and the biological behavior of the cell. By analyzing and comparing the protein patterns of different cells, it is possible to recognize the cell type and to identify the most typical features of these cells. In applied pathology it is

HBc3.6 were labeled separately with Cy3 (*red*) and Cy5 (*blue*), respectively.

**2.6.11 2-DE protein pattern in classification of carcinoma cells** 

labeled with Cy3 (*red*) and HBc3.6 lysate labeled with Cy5 (*blue*) (*left panel*).

decreased expression of cytokeratin and tropomyosin polypeptides can be discerned (Fig. 9 & 10). (Franzen, *et al.* 1997 *Electrophoresis*, 18 : 582–587.).

Fig. 8. Protein expression comparisons of normal samples and SCC samples. Up- and downregulated proteins (14-3-3ε, annexin A1, and SCCA-2) were selected and magnified gel images were presented (A). From the PDQuest 2-D software quantification, the expression difference was statistically meaningful (*P* value < 0.05) (B). (*Source: Bae et al., 2005 Gynecologic Oncol., 99: 26-35)*

The same gel was post-stained with SyproRuby dye (*right panel*). 100 µg of each lysate from serum-starved cells were analyzed on a 9–16% gradient gel. *Circles* represent differentially expressed proteins detectable by both methods. *Arrows* represent spots detected by SyproRuby but not Cy dye labeling. *B*, the shift in molecular weight between the modified and unmodified proteins was visualized by image overlaying. The DIGE image (*Blue*) was overlaid with the SYPRO image (*Red*).

decreased expression of cytokeratin and tropomyosin polypeptides can be discerned (Fig. 9

Fig. 8. Protein expression comparisons of normal samples and SCC samples. Up- and downregulated proteins (14-3-3ε, annexin A1, and SCCA-2) were selected and magnified gel images were presented (A). From the PDQuest 2-D software quantification, the expression

The same gel was post-stained with SyproRuby dye (*right panel*). 100 µg of each lysate from serum-starved cells were analyzed on a 9–16% gradient gel. *Circles* represent differentially expressed proteins detectable by both methods. *Arrows* represent spots detected by SyproRuby but not Cy dye labeling. *B*, the shift in molecular weight between the modified and unmodified proteins was visualized by image overlaying. The DIGE image (*Blue*) was

difference was statistically meaningful (*P* value < 0.05) (B). (*Source: Bae et al., 2005* 

*Gynecologic Oncol., 99: 26-35)*

overlaid with the SYPRO image (*Red*).

& 10). (Franzen, *et al.* 1997 *Electrophoresis*, 18 : 582–587.).

Fig. 9. Sensitivity of 2D-DIGE and compatibility with SYPRO gel staining. *A*, comparison of 2D-DIGE imaging and SyproRuby poststaining. Merged Cy dye image of HB4a lysate labeled with Cy3 (*red*) and HBc3.6 lysate labeled with Cy5 (*blue*) (*left panel*).

Fig. 10. 2D-DIGE is a reproducible detection method. Duplicate samples of HB4a and HBc3.6 were labeled separately with Cy3 (*red*) and Cy5 (*blue*), respectively.
