Hindawi Publishing Corporation

Journal of Biomedicine and Biotechnology

Volume 2011, Article ID 193052, 8 pages
doi:10.1155/2011/193052

Research Article
Separation and Identification of HSP-Associated
Protein Complexes from Pancreatic Cancer Cell Lines Using 2D
CN/SDS-PAGE Coupled with Mass Spectrometry

Zhiyun Zhao, Hui Liu, Xinli Wang, Xiaodong Wang, and Zhili Li
Department of Biophysics and Structural Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School
of Basic Medicine, Peking Union Medical College, Beijing 100005, China

Correspondence should be addressed to Zhili Li, [email protected]

Received 16 May 2011; Revised 12 August 2011; Accepted 17 August 2011

Academic Editor: Kapil Mehta

Copyright © 2011 Zhiyun Zhao et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Protein complexes are a cornerstone of many biological processes and together they form various types of molecular machinery. A
broad understanding of these protein complexes is crucial for revealing and building models of protein function and regulation.
Pancreatic cancer is a highly lethal disease which is difficult to diagnose at early stage and even more difficult to cure. In this
study, we applied a gradient clear native gel system combined with subsequent second-dimensional SDS-PAGE to separate protein
complexes from cell lysates of SW1990 and PANC-1 pancreatic cancer cell lines with different degrees of differentiation. Ten
heat-shock-protein- (HSP-) associated protein complexes were separated and identified, and the differentially expressed proteins
related to cancers were also found, such as HSP60, protein disulfide-isomerase A4 (ERp72), and transitional endoplasmic reticulum
ATPase (TER ATPase).

1. Introduction in-gel catalytic activity assays and analyses of fluorescent-

labeled proteins [10, 11]. After BN/CN-PAGE, a second-di-
Most proteins exert their functions by stable or transient in- mensional SDS-PAGE can be performed to separate polypep-
teractions with other proteins either as homo- or heter- tides as components of protein complexes [12]. Previous
ooligomeric protein complexes [1, 2]. The diversity of studies using BN/CN-PAGE approach mainly focused on
biological processes is derived from the dynamic network membrane, chloroplast, mitochondria, and plasma protein
of protein interactions in cell. These interactions form the complexes [13–18]. So far, limited studies were associated
basis of the quaternary structure of multimeric proteins with the protein complexes from cell lysate [19, 20].
and represent one of the most complex levels of structural Pancreatic cancer is a malignant neoplasm of the
organization in biological molecules [3, 4]. A broad under- pancreas. During the past four decades, the incidence of
standing of protein complexes and protein interactions is pancreatic cancer has risen steadily [21]. The prognosis is
crucial for revealing and building models of protein function relatively poor with less than 5% five-year survival rate, and
and regulation. complete remission is rare [22].
A variety of approaches were used to study protein- Heat shock proteins (HSPs) are a highly conserved family
protein interactions, such as tandem affinity purification of proteins among living organisms during evolution [23].
(TAP) [5], coimmunoprecipitation (co-IP) [6], yeast two- A cell may have a variety of HSPs in cellular compartments
hybrid assay [7], blue/clear (colorless) native PAGE (BN/CN- such as endoplasmic reticulum, mitochondria, and cytosol
PAGE) [8], and other gel approaches [9]. Among these [24]. HSPs have multiple functions such as proper folding
methods, BN/CN-PAGE is widely used [2] and CN-PAGE of nascent chains, protein translocation across membranes,
is milder than BN-PAGE and offers special advantages for protein disaggregation, preservation and restructuring of the
2 Journal of Biomedicine and Biotechnology

cytoskeleton, and targeting damaged proteins for degrada- bands or lanes were then dipped into equilibrating buffer
tion [24, 25]. HSPs may serve as potential molecular targets B supplemented with 62.5 mM Tris-HCl pH 6.8, 2% SDS,
for therapeutic intervention in the treatment of diseases, 10% glycerol, and 2.5% iodoacetamide for 20 min at room
such as cancer, Alzheimer’s, and Parkinson’s diseases [26]. temperature. The bands or lanes were washed with deionized
The heat shock protein peptide complex-96-based vaccines water followed by 12% SDS-PAGE separation. Gels were
in melanoma were also tested in phase III clinical trials in stained with Coomassie blue G-250.
melanoma and kidney cancer [27]. In pancreatic cancer, it
was found that HSP70 and HSP90 were overexpressed [28– 2.5. In-Gel Digestion and Protein Identification. Protein
30]. bands or spots from the second-dimensional SDS-PAGE
In this study, we applied a gradient clear native gel fol- were excised and cut into small pieces. After destained, re-
lowed by a second-dimensional SDS-PAGE to reveal the duced, and alkylated, the gel pieces were dehydrated in ac-
HSP-associated protein complexes from the cell lysates of etonitrile and covered by some volume of 12.5 ng/μL trypsin
SW1990 and PANC-1 pancreatic cell lines. The components in 25 mM ammonium bicarbonate for 1 h at 4◦ C. Protein
of these complexes were then identified by MALDI-MS and digestion was performed for overnight at 37◦ C.
MS/MS. 1 μL of the digest was spotted onto an MTP AnchorChip
384/400 plate and dried at room temperature before the
2. Materials and Methods addition of 1 μL matrix solution of 1 mg/mL CHCA in
33% ACN/0.1% TFA. After the mixtures were completely
2.1. Materials and Chemicals. Protease inhibitor cocktail tab- dried at room temperature, the AnchorChip was delivered
lets were obtained from Roche Applied Science (Indianapo- into autoflex III MALDI-TOF/TOF MS (Bruker Daltonics,
lis, Ind, USA). Sequencing-grade trypsin was purchased Billerica, Mass, USA). The mass spectrometer was operated
from Roche diagnostics (Mannheim, Germany). α-cyano-4- under 20 kV accelerating voltage in the positive-ion reflec-
hydroxycinnamic acid (CHCA) was from Sigma-Aldrich (St. tron mode with the mass range of m/z 800–4000.
Louis, Mo, USA). All other chemicals were obtained from The monoisotopic peptide mass was generated by Flex-
Merck (Darmstadt, Germany). analysis 3.0 (Bruker Daltonics), and the database search
was performed by Biotools 3.1 software (Bruker Daltonics)
2.2. Sample Preparation. PANC-1 and SW1990 were cultured against Swiss-Prot database using the MASCOT search
in Dulbecco’s Modified Eagle’s medium (DMEM) and algorithm. The parameters are as follows: one missed tryptic
RPMI-1640 medium, respectively, supplemented with 10% cleavage; taxonomy: Homo sapiens; carbamidomethylation
fetal calf serum and 100 U/mL penicillin/streptomycin at of cysteine as fixed modification; oxidation of methionine
37◦ C and 5% CO2 in a humidified atmosphere. and N-terminal pyroglutamylation as variable modifications;
After harvested cells were washed with PBS, the cells were mass tolerance: 0.1 Da for precursor ions and 0.5 Da for
lysed by three-cycle liquid N2 /4◦ C in lysis buffer (50 mM product ions. Results were considered as significant with
Tris-HCl, pH 7.5, 5 mM MgCl2 , 150 mM NaCl, 0.05% NP- MASCOT score higher than 60 (P < 0.05). Sequence
40, 1 mM DTT, 2 × Roche protease inhibitor cocktail, 10 mM coverage, number of matched MS/MS, and the experimental
sodium fluoride, and 10 mM sodium pyrophosphate). The molecular weight based on SDS-PAGE were also used to
lysate was centrifuged at 20, 000 xg for 60 min at 4◦ C. The confirm further identification.
supernatant containing protein complexes was centrifuged
again at 10, 000 xg for 30 min at 4◦ C for at least three times to 2.6. Protein-Protein Interaction Analysis. A protein-protein
remove the lipids in the lysate. The protein concentration was interaction network of the proteins identified in the HSP-
determined by Bradford assay using bovine serum albumin associated complexes was created using STRING database
as the standard protein. (http://string-db.org/) through inputting the Swiss-Prot ac-
cession numbers.
2.3. Separation of HSP-Associated Protein Complexes. CN-
PAGE was performed based on previous protocols [31, 32].
100 μg of proteins was loaded onto the gel. CN-PAGE was
3. Results and Discussion
performed with 4% stacking gel and 6%, 5–10% or 5–12% 3.1. Separation of Protein Complexes by CN-PAGE. Due to
separating gel with 13 cm casting gel system, respectively. GE the aberrant effect of hydrophobic lipids in lysate on CN-
Healthcare HMW-Native protein markers (GE Healthcare, PAGE performance, several approaches have been tried and
Uppsala, Sweden) were used. The gel was run at 4◦ C until finally the centrifugation was chosen to remove lipids. It
the xylene cyanol reached the gel bottom. should be noted that at least three-time centrifugations
were needed for complete removal of lipids. To establish
2.4. Separation of the Components of HSP-Associated Protein the conditions of CN-PAGE for adequately resolving protein
Complexes. For further separation in subsequent second- complexes, three separating gels were tried: 6% nongradient
dimensional SDS-PAGE, the protein complex bands or the (left, Figure 1(a)), 5–10% gradient (middle, Figure 1(a))
whole lanes from CN-PAGE were cut out and equilibrated and 5–12% gradient gels (right, Figure 1(a)). As shown in
for 20 min in equilibrating buffer A containing 62.5 mM Figure 1(a), protein complexes were well separated on 5–12%
Tris-HCl pH 6.8, 2% SDS, 10% glycerol, and 1% DTT. The gradient gel compared to nongradient and 5–10% gel.
Journal of Biomedicine and Biotechnology 3

1 2 3
M S M S M S
(kDa) (kDa) (kDa)
669
669 669 A B C D E F G H I J
A
440 B
2 2
440
440 3 3 9 9 9 9
4 4 4 4 4 4 4
C 5 5 5 5 5 5 5 5
232 D 6 6 6 6 6 6
232 E 10
1 1 1 1 1 1
140 232 7 7 7 7 7 7
F
140 G 8 8 8 8 8
H 13
11 14
140 I
J
12

66
66 66 15

(a) (b)

Figure 1: 6% (1), 5–10% (2), and 5–12% (3) separating gels were used to separate the protein complexes from SW1990 cell lysate. M: GE
Healthcare HMW-Native protein markers; S: cell lysate of SW1990 (a). SDS-PAGE of ten protein complexes labeled with A–J from 5–12%
CN-PAGE. At least two-band combination of each complex was used to run SDS-PAGE (b).

Table 1: Identification results of HSP-associated protein complexes.

C D E F G H I J
ORP-150 ORP-150 Alpha-actinin-4 Alpha-actinin-4 Alpha-actinin-4 Alpha-actinin-4 — —
— HSP90 HSP90 HSP90 HSP90 HSP90 HSP90 HSP90
— HSP90-beta HSP90-beta HSP90-beta HSP90-beta HSP90-beta HSP90-beta HSP90-beta
BiP BiP BiP BiP BiP BiP BiP BiP
HSP71 HSP71 HSP71 HSP71 HSP71 HSP71 — Beta-actin
ERp57 ERp57 ERp57 ERp57 ERp57 ERp57 — Gamma-actin
— ERp5 ERp5 ERp5 ERp5 ERp5 — PGAM-B
GRP-94 GRP-94 SAHase HSP60 HSP60 HSP60 HSP60 —
— NAD-ME — LDH-A — — — —

3.2. Identification and Interaction Analysis of HSP-Associated while the BN-PAGE migration distance depends only on the
Protein Complexes. The protein complexes in the CN-PAGE pore size of the gel.
(right, Figure 1(a)) were individually separated by second- For complex C, the most intensively stained protein was
dimensional SDS-PAGE (Figure 1(b)). The bands in the identified as endoplasmin (GRP-94) (band 3, Figure 1(b)),
SDS-PAGE were excised, digested, and identified by MALDI- which is involved in the processing and transporting of
MS and MS/MS. The identified proteins are listed in Table S1 secreted proteins [34]. The other proteins are hypoxia
and their corresponding MASCOT score, sequence coverage, upregulated protein 1 (ORP-150), 78 kDa glucose-regulated
and number of matched MS/MS are also listed in Table S1 protein (BiP), HSP71, and protein disulfide-isomerase A3
(see Table S1 in supplementary material available online at (ERp57) (Table 1). GRP-94, OPR-150, and BiP are glucose-
doi:10.1155/2011/193052). The components of each com- regulated proteins. GRP-94 and BiP are also members of
plex are shown in Table 1. the 90 and 70 kDa families of HSPs, and OPR-150 is a large
The apparent masses of complex A and B are about HSP70-, HSP110-like protein of the endoplasmic reticulum
470 kDa (right, Figure 1(a)), and the components of both [35]. HSP71 belongs to the heat shock protein 70 family.
complexes were identified as HSP60 (band 1, Figure 1(b)). ERp57 is a member of the protein-disulfide-isomerase-
A previous study showed that the HSP60 complex is a (PDI-) like family, and it has been implicated in human
homoheptamer with apparent mass of 420 kDa in BN-PAGE pathologies including cancer and Alzheimer’s disease [36].
[33]. The mass difference may be due to the fact that the CN- In addition to the proteins identified in complex C, four
PAGE migration distance depends on not only the pore size other proteins were identified in complex D. They are heat
of gradient gel but also the protein intrinsic charge and size, shock protein HSP 90-alpha (HSP90), heat shock protein
4 Journal of Biomedicine and Biotechnology

α-actinin-4

Γ-actin

β-actin

ORP-150

ERp57

HSP71
HSP90
GRP-94

LDH-A
HSP90β
BiP
SAHase NAD-ME

ERp5 HSP60

PGAM-B

(a)
E
SAHase
F
C
D LDH-A
ERp57 α-actinin-4
GRP-94 HSP71 ERp5
G J
NAD-ME HSP90
HSP90β I
H β-actin
ORP-150 BiP Γ-actin
PGAM-B

HSP60

(b)

Figure 2: Interaction analysis of proteins from the HSP-associated complexes using the STRING database. The lines represent interactions
and individual proteins are depicted as nodes. Stronger interactions are represented by thicker lines (a). The HSP-associated complexes from
this study are shown in different ellipses. The sign of each protein complex is indicated on the edge of each ellipse (b).

HSP90-beta (HSP90-beta), protein disulfide isomerase P5 function of numerous oncoproteins [37, 38]. ERp5 as a
(ERp5), and NAD-dependent malic enzyme (NAD-ME). member of the protein-disulfide-isomerase- (PDI-) like fam-
HSP90 and HSP90-beta belong to the heat shock protein ily was found to interact with BiP [39]. NAD-ME plays an
90 family. HSP90 proteins play essential housekeeping func- important role in the metabolism of glutamine for energy
tions, and they are used by cancer cells to facilitate the production in rapidly proliferating tissues and tumors [40].
Journal of Biomedicine and Biotechnology 5

S CN-PAGE P CN-PAGE
(kDa) S P
669 (kDa)
A+B 1 170
1 3 130
440
100
2 2 70
4 4 5
SDS-PAGE
232 55

F 40
140
35

K 25

66

(a) (b)

Figure 3: The protein complexes differentially expressed between SW1990; and PANC-1 cell lines were labeled by A+B, F, and K in CN-
PAGE (a). The proteins differentially expressed between two cell lines in the second-dimensional SDS-PAGE were labeled in number (b) S:
cell lysate of SW1990; P: cell lysate of PANC-1.

Table 2: Differential protein complexes and differential proteins of complex J are HSP90, HSP90-beta, BiP, beta-actin, gam-
between SW1990 and PANC-1 cell lines. ma-actin, and phosphoglycerate mutase 1 (PGAM-B). Actin
Spot number (protein name; fold is one of the most highly conserved proteins. The beta- and
Complex (fold change SW1990 gamma-actins co-exist in most cell types as components of
change SW1990 to PANC-1) in
to PANC-1) in Figure 3(a) the cytoskeleton and mediators of internal cell motility [44].
Figure 3(b)
Complex (A+B) (1.61) 2 (HSP60; 1.85) PGAM-B is a key enzyme of the glycolytic pathway, which
converts 3-phosphoglycerate to 2-phosphoglycerate [45].
Complex K (1.96) 4 (ERp72; 2.11)
A network of the identified proteins in the HSP-as-
Complex F (found only in
— sociated complexes was obtained using the STRING software
SW1990)
(Figure 2(a)). In this network all proteins interact with other
— 1 (TER ATPase; 1.52) proteins except SAHase. Previously, it was also found that
3 (Transglutaminase-2; found HSP90, HSP70, and HSP60 interacted with each other in
—
only in PANC-1) human cells [19, 46]. The HSP-associated complexes were
5 (Annexin A6; found only in plotted in Figure 2(b). These complexes were shown in differ-
—
PANC-1) ent ellipses, respectively, indicating that HSP90, HSP90-beta,
and HSp71 are “hot” proteins in these complexes. The major
HSPs have five conserved classes: HSP100, HSP90, HSP70,
The proteins identified in complex E are alpha-actinin-4, HSP60, and the small heat shock proteins (sHSPs) [47].
HSP90, HSP90-beta, BiP, HSP71, ERp57, ERp5, and adeno- Each chaperone class displays relatively specialized functions
sylhomocysteinase (SAHase). Except alpha-actinin-4 and but the members of different classes often work together
SAHase, the rest also were found in the complex D. Alpha- to perform their duties in the form of larger multi-protein
actinin-4 regulates the actin cytoskeleton and is associated
complexes [24, 48]. Our results indicate that, in pancreatic
with cell motility and cancer invasion [41]. SAHase may play
cancer cells, HSP90, HSP90-beta, and HSP71 could form
a key role in the control of methylations via regulation of the
many different complexes with other proteins.
intracellular concentration of adenosylhomocysteine [42].
The identified components of complexes of F, G, and
H are similar. Alpha-actinin-4, HSP90, HSP90-beta, BiP, 3.3. Protein Complexes Expression Differences between
HSP71, ERp57, ERp5, and HSP60 were found in these three SW1990 and PANC-1 Cell Lines. SW1990 and PANC-1 are
complexes. It should be noted that HSP60 presents in these pancreatic cancer cell lines derived from pancreatic duct
three complexes, although not in the complexes of C, D, and with different differentiation degrees. SW1990 is a well to
E. HSP60 is a molecular chaperone known to assist protein moderately differentiated cell line established from a spleen
folding in prokaryotes and in eukaryotic cell organelles. Its metastasis of pancreatic duct cancer, and PANC-1 is a poorly
roles in human cancer development are currently actively differentiated cell line [49, 50]. The spots with differential
being investigated [43]. expression over 1.5-folds were regarded as candidates of
The identified components of complex I are HSP90, differential expression, followed by MALDI-MS and MS/MS
HSP90-beta, BiP, and HSP60, and the identified components identifications (Table S2).
6 Journal of Biomedicine and Biotechnology

The expression differences of the protein complexes la- suppressor activity via facilitating the Ca2+ -dependent mem-
beled by A+B, F, and K between SW1990 and PANC-1 cell brane targeting of p120GAP to downregulate Ras activity
lines are shown in Figure 3(a), and differential expression [58].
proteins labeled in number are shown in Figure 3(b).
Table 2 summarized the link between protein complexes
in Figure 3(a) and spots in Figure 3(b) and also provided 4. Conclusions
the fold changes of these complexes or individual protein
between SW1990 and PANC-1 cell lines. Compared to In this study, protein complexes in SW1990 and PANC-1 cell
PANC-1, the complexes (A+B) (A+B means the combination lysates were separated and identified by 2D CN/SDS-PAGE
of complexes A and B), F, and K were upexpressed in coupled with mass spectrometry. Ten HSP-associated com-
SW1990 (Figure 3(a)). The expression trends of the com- plexes were identified. Our study indicates that HSP90,
plexes (A+B) and K in Figure 3(a) were in accordance with HSP90-beta, and HSP71 may be “core” proteins in these
those of their components corresponding to spots 2 and 4, complexes. The differentially expressed proteins such as
respectively (Figure 3(b)). The components of the complex HSP60, TER ATPase, and transglutaminase-2 also were pro-
F were not, however, enough in abundance to be observed filed in SW1990 and PANC-1 cells, which are cancer-related
in the SDS-PAGE. Additionally, TER ATPase (spot 1, proteins. It should be noted that the 2D CN/SDS-PAGE
Figure 3(b)), protein-glutamine gamma-glutamyltransferase coupled with mass spectrometry strategy may be a simple
2 (transglutaminase-2, spot 3, Figure 3(b)), and annexin A6 and powerful tool to screen protein-protein interactions
(spot 5, Figure 3(b)) were obviously observed in the SDS- among different cell types and tissues.
PAGE.
As shown in Figure 3(b), TER ATPase (spot 1) with many
important biological functions [51] was upexpressed in Abbreviations
SW1990 cells, which is associated with intestinal tumori- BN-PAGE: blue native PAGE.
genesis as indicated in previous study [52]. HSP60 (spot CN-PAGE: clear (colorless) native PAGE.
2, Figure 3(b)) was upexpressed in SW1990 cells. Although HSP: heat shock protein.
the role of HSP60 in cancer has not been determined yet, HPRD: human protein reference database.
upregulation or downregulation of this chaperone has been
reported in various tumors, histopathological diagnosis and
prognosis assessment [43, 53]. HSP60 was found to be Acknowledgments
downregulated in tumors compared with normal pancreas
tissues in the 2-DE analysis; on the contrary, the data from This investigation was supported by Grant 21075137 from
immunohistochemistry showed that HSP60 was strongly the National Natural Science Foundation of China and Grant
expressed in both normal acinar cells and small ducts in 2006AA02Z154 awarded by National High-Tech R&D Pro-
normal pancreas tissues and tumor cells in all the pancreatic gram of China (863 Program). The authors have no conflict
ductal adenocarcinoma tissues [54]. of interests to declare.
The complex F was found only in SW1990 cell lysate
(Figure 3(a)). The difference of this HSP-associated complex
in the two pancreatic cancer cells may suggest the importance References
of HSPs in cancers as other studies reported [23, 48].
[1] M. Abu-Farha, F. Elisma, and D. Figeys, “Identification
The main function of transglutaminase-2 (spot 3, of protein-protein interactions by mass spectrometry coupled
Figure 3(b)) is to catalyze the cross-linking of proteins and techniques,” Advances in Biochemical Engineering/Biotechnol-
conjugating of polyamines to proteins, and its expression ogy, vol. 110, pp. 67–80, 2008.
was elevated in most pancreatic ductal adenocarcinoma [2] F. Krause, “Detection and analysis of protein-protein interac-
tumors and cell lines [55]. In melanoma, transglutaminase-2 tions in organellar and prokaryotic proteomes by native gel
absence in the host is a favoring condition for the formation electrophoresis: (Membrane) protein complexes and super-
and development of the metastasis, while the presence of complexes,” Electrophoresis, vol. 27, no. 13, pp. 2759–2781,
transglutaminase-2 in the tumor cells might be requested for 2006.
the development of the metastasis [56], which is consistent [3] A. J. Link, T. C. Fleischer, C. M. Weaver, V. R. Gerbasi, and
with our finding of transglutaminase-2 expression only in J. L. Jennings, “Purifying protein complexes for mass spec-
the poorly differentiated PANC-1 cells. Protein disulfide- trometry: applications to protein translation,” Methods, vol.
isomerase A4 (ERp72) (complex K, Figure 3(a); spot 4, 35, no. 3, pp. 274–290, 2005.
[4] R. P. Bahadur and M. Zacharias, “The interface of protein-
Figure 3(b)) is another protein that was upexpressed in the
protein complexes: analysis of contacts and prediction of
well to moderately differentiated and metastatic SW1990 cell interactions,” Cellular and Molecular Life Sciences, vol. 65, no.
line. In hepatocellular carcinoma (HCC), the upregulation of 7-8, pp. 1059–1072, 2008.
ERp72 was discovered not only in highly metastatic HCC cell [5] X. Xu, Y. Song, Y. Li, J. Chang, H. Zhang, and L. An, “The
lines but also in highly metastatic and already metastasized tandem affinity purification method: an efficient system for
HCC patient’s sera [57]. Annexin A6 (spot 5, Figure 3(b)), protein complex purification and protein interaction iden-
which was upregulated in PANC-1 cells, regulates the release tification,” Protein Expression and Purification, vol. 72, no. 2,
of Ca2+ from intracellular stores, and it displays tumor pp. 149–156, 2010.
Journal of Biomedicine and Biotechnology 7

[6] E. A. Elion, “Detection of protein-protein interactions by co- [22] P. Ghaneh, E. Costello, and J. P. Neoptolemos, “Biology
precipitation,” Current Protocols in Neuroscience, 2006, chapter and management of pancreatic cancer,” Postgraduate Medical
5, Unit 5.25. Journal, vol. 84, no. 995, pp. 478–497, 2008.
[7] B. Suter, S. Kittanakom, and I. Stagljar, “Two-hybrid technolo- [23] V. Dudeja, S. M. Vickers, and A. K. Saluja, “The role of heat
gies in proteomics research,” Current Opinion in Biotechnology, shock proteins in gastrointestinal diseases,” Gut, vol. 58, no. 7,
vol. 19, no. 4, pp. 316–323, 2008. pp. 1000–1009, 2009.
[8] I. Wittig and H. Schägger, “Native electrophoretic techniques [24] L. Tutar and Y. Tutar, “Heat shock proteins; An overview,”
to identify protein-protein interactions,” Proteomics, vol. 9, no. Current Pharmaceutical Biotechnology, vol. 11, no. 2, pp. 216–
23, pp. 5214–5223, 2009. 222, 2010.
[9] X. Wang, G. Chen, H. Liu, Z. Zhao, and Z. Li, “Four-di- [25] A. L. Joly, G. Wettstein, G. Mignot, F. Ghiringhelli, and C.
mensional orthogonal electrophoresis system for screening Garrido, “Dual role of heat shock proteins as regulators of
protein complexes and protein-protein interactions combined apoptosis and innate immunity,” Journal of Innate Immunity,
with mass spectrometry,” Journal of Proteome Research, vol. 9, vol. 2, no. 3, pp. 238–247, 2010.
no. 10, pp. 5325–5334, 2010. [26] V. V. Malaitsev, I. M. Bogdanova, and O. V. Makarova,
[10] I. Wittig and H. Schägger, “Advantages and limitations of “Heat shock proteins and their role in the development of
clear-native PAGE,” Proteomics, vol. 5, no. 17, pp. 4338–4346, pathological processes,” Arkhiv Patologii, vol. 70, no. 6, pp. 31–
2005. 38, 2008.
[11] I. Wittig and H. Schägger, “Features and applications of blue- [27] A. Di Pietro, G. Tosti, P. F. Ferrucci, and A. Testori, “Heat shock
native and clear-native electrophoresis,” Proteomics, vol. 8, no. protein peptide complex 96-based vaccines in melanoma: how
19, pp. 3974–3990, 2008. far we are, how far we can get,” Human Vaccines, vol. 5, no. 11,
[12] F. Krause and H. Seelert, “Detection and analysis of protein- pp. 727–737, 2009.
protein interactions of organellar and prokaryotic proteomes [28] Y. Oki and A. Younes, “Heat shock protein-based cancer
by blue native and colorless native gel electrophoresis,” Current vaccines,” Expert Review of Vaccines, vol. 3, no. 4, pp. 403–411,
Protocols in Protein Science, 2008, chapter 14, unit 14.11. 2004.
[13] V. Reisinger and L. A. Eichacker, “Analysis of membrane [29] A. Saluja and V. Dudeja, “Heat shock proteins in pancreatic
protein complexes by blue native PAGE,” Proteomics, vol. 1, diseases,” Journal of Gastroenterology and Hepatology, vol. 23,
supplement 2, pp. 6–15, 2006. supplement 1, pp. S42–S45, 2008.
[14] M. Kügler, L. Jänsch, V. Kruft, U. K. Schmitz, and H. P. [30] T. M. Gress, F. Müller-Pillasch, C. Weber et al., “Differential
Braun, “Analysis of the chloroplast protein complexes by expression of heat shock proteins in pancreatic carcinoma,”
blue-native polyacrylamide gel electrophoresis (BN-PAGE),” Cancer Research, vol. 54, no. 2, pp. 547–551, 1994.
Photosynthesis Research, vol. 53, no. 1, pp. 35–44, 1997. [31] S. Elsasser, M. Schmidt, and D. Finley, “Characterization of
[15] L. G. J. Nijtmans, N. S. Henderson, and I. J. Holt, “Blue Native the proteasome using native gel electrophoresis,” Methods in
electrophoresis to study mitochondrial and other protein Enzymology, vol. 398, pp. 353–363, 2005.
complexes,” Methods, vol. 26, no. 4, pp. 327–334, 2002. [32] G. Chen, Y. Luo, X. Wang et al., “A relatively simple and
[16] P. S. Brookes, A. Pinner, A. Ramachandran et al., “High economical protocol for proteomic analyses of human 20S
throughput two-dimensional blue-native electrophoresis: a proteasome: compatible with both scaled-up and scaled-down
tool for functional proteomics of mitochondria and signaling purifications,” Electrophoresis, vol. 30, no. 14, pp. 2422–2430,
complexes,” Proteomics, vol. 2, no. 8, pp. 969–977, 2002. 2009.
[17] T. Manabe and Y. Jin, “Noncovalent interactions in human [33] N. H. Reifschneider, S. Goto, H. Nakamoto et al., “Defining
plasma proteins analyzed by the comparison of nondena- the mitochondrial proteomes from five rat organs in a
turing and denaturing micro-2-D gel electrophoresis pat- physiologically significant context using 2D blue-native/SDS-
terns after polypeptide assignment using matrix-assisted laser PAGE,” Journal of Proteome Research, vol. 5, no. 5, pp. 1117–
desorption/ionization-mass spectrometry and peptide mass 1132, 2006.
fingerprinting,” Electrophoresis, vol. 29, no. 12, pp. 2672–2688, [34] J. C. Christianson, T. A. Shaler, R. E. Tyler, and R. R.
2008. Kopito, “OS-9 and GRP94 deliver mutant α1-antitrypsin to
[18] S. Sunderhaus, H. Eubel, and H. P. Braun, “Two-dimensional the Hrd1?SEL1L ubiquitin ligase complex for ERAD,” Nature
blue native/blue native polyacrylamide gel electrophoresis for Cell Biology, vol. 10, no. 3, pp. 272–282, 2008.
the characterization of mitochondrial protein complexes and [35] X. Chen, D. Eastonb, H.-J. Oh, D.-S. Lee-Yoon, X. Liu, and
supercomplexes,” Methods in Molecular Biology, vol. 372, pp. J. Subjeck, “The 170 kDa glucose regulated stress protein is
315–324, 2007. a large HSP70-, HSP110-like protein of the endoplasmic
[19] K. Liu, L. Qian, J. Wang et al., “Two-dimensional blue reticulum,” FEBS Letters, vol. 380, no. 1-2, pp. 68–72, 1996.
native/SDS-PAGE analysis reveals heat shock protein chap- [36] H. Coe and M. Michalak, “ERp57, a multifunctional endoplas-
erone machinery involved in hepatitis B virus production in mic reticulum resident oxidoreductase,” International Journal
HepG2.2.15 cells,” Molecular and Cellular Proteomics, vol. 8, of Biochemistry and Cell Biology, vol. 42, no. 6, pp. 796–799,
no. 3, pp. 495–505, 2009. 2010.
[20] M. M. Camacho-Carvcajal, B. Wollscheid, R. Aebersold, V. [37] J. Trepel, M. Mollapour, G. Giaccone, and L. Neckers, “Tar-
Steimle, and W. W. A. Schamel, “Two-dimensional Blue geting the dynamic HSP90 complex in cancer,” Nature Reviews
Native/SDS gel electrophoresiss of multi-protein complexes Cancer, vol. 10, no. 8, pp. 537–549, 2010.
from whole cellular lysates: a proteomics approach,” Molecular [38] H. Hao, Y. Naomoto, X. Bao et al., “HSP90 and its inhibitors,”
and Cellular Proteomics, vol. 3, no. 2, pp. 176–182, 2004. Oncology Reports, vol. 23, no. 6, pp. 1483–1492, 2010.
[21] A. Jemal, R. Siegel, E. Ward et al., “Cancer statistics, 2006,” [39] C. E. Jessop, R. H. Watkins, J. J. Simmons, M. Tasab, and
CA-A Cancer Journal for Clinicians, vol. 56, no. 2, pp. 106–130, N. J. Bulleid, “Protein disulphide isomerase family members
2006. show distinct substrate specificity: P5 is targeted to BiP client
8 Journal of Biomedicine and Biotechnology

proteins,” Journal of Cell Science, vol. 122, no. 23, pp. 4287– [54] T. Qi, J. Han, Y. Cui, M. Zong, X. Liu, and B. Zhu, “Com-
4295, 2009. parative proteomic analysis for the detection of biomarkers
[40] Z. Yang, C. W. Lanks, and L. Tong, “Molecular mechanism for in pancreatic ductal adenocarcinomas,” Journal of Clinical
the regulation of human mitochondrial NAD(P)+-Dependent Pathology, vol. 61, no. 1, pp. 49–58, 2008.
malic enzyme by ATP and umarate,” Structure, vol. 10, no. 7, [55] K. Mehta, “Biological and therapeutic significance of tissue
pp. 951–960, 2002. transglutaminase in pancreatic cancer,” Amino Acids, vol. 36,
[41] K. Honda, T. Yamada, R. Endo et al., “Actinin-4, a novel no. 4, pp. 709–716, 2009.
actin-bundling protein associated with cell motility and cancer [56] G. Di Giacomo, A. Lentini, S. Beninati, M. Piacentini, and
invasion,” Journal of Cell Biology, vol. 140, no. 6, pp. 1383– C. Rodolfo, “In vivo evaluation of type 2 transglutaminase
1393, 1998. contribution to the metastasis formation in melanoma,”
[42] X. Yang, Y. Hu, D. H. Yin et al., “Catalytic strategy of Amino Acids, vol. 36, no. 4, pp. 717–724, 2009.
S-Adenosyl-L-homocysteine hydrolase: transition-state stabi- [57] N. Chen, W. Sun, X. Deng et al., “Quantitative proteome
lization and the avoidance of abortive reactions,” Biochemistry, analysis of HCC cell lines with different metastatic potentials
vol. 42, no. 7, pp. 1900–1909, 2003. by SILAC,” Proteomics, vol. 8, no. 23-24, pp. 5108–5118, 2008.
[43] F. Cappello, E. C. de Macario, L. Marasà, G. Zummo, and A. [58] T. Grewal, M. Koese, C. Rentero, and C. Enrich, “Annexin A6-
J. Macario, “Hsp60 expression, new locations, functions and regulator of the EGFR/Ras signalling pathway and cholesterol
perspectives for cancer diagnosis and therapy,” Cancer Biology homeostasis,” International Journal of Biochemistry and Cell
& Therapy, vol. 7, no. 6, pp. 801–809, 2008. Biology, vol. 42, no. 5, pp. 580–584, 2010.
[44] D. Tondeleir, D. Vandamme, J. Vandekerckhove, C. Ampe,
and A. Lambrechts, “Actin isoform expression patterns during
mammalian development and in pathology: insights from
mouse models,” Cell Motility and the Cytoskeleton, vol. 66, no.
10, pp. 798–815, 2009.
[45] F. Ren, H. Wu, Y. Lei et al., “Quantitative proteomics identi-
fication of phosphoglycerate mutase 1 as a novel therapeutic
target in hepatocellular carcinoma,” Molecular Cancer, vol. 9,
article 81, 2010.
[46] M. Nakamura, H. Morisawa, S. Imajoh-Ohmi, C. Taka-
mura, H. Fukuda, and T. Toda, “Proteomic analysis of pro-
tein complexes in human SH-SY5Y neuroblastoma cells by
using blue-native gel electrophoresis: an increase in lamin
A/C associated with heat shock protein 90 in response to
6-hydroxydopamine-induced oxidative stress,” Experimental
Gerontology, vol. 44, no. 6-7, pp. 375–382, 2009.
[47] H. P. Kim, D. Morse, and A. M. K. Choi, “Heat-shock proteins:
new keys to the development of cytoprotective therapies,”
Expert Opinion on Therapeutic Targets, vol. 10, no. 5, pp. 759–
769, 2006.
[48] E. M. Karapanagiotou, K. Syrigos, and M. W. Saif, “Heat
shock protein inhibitors and vaccines as new agents in cancer
treatment,” Expert Opinion on Investigational Drugs, vol. 18,
no. 2, pp. 161–174, 2009.
[49] A. P. Kyriazis, W. B. McCombs, and A. A. Sandberg,
“Establishment and characterization of human pancreatic
adenocarcinoma cell line SW-1990 in tissue culture and the
nude mouse,” Cancer Research, vol. 43, no. 9, pp. 4393–4401,
1983.
[50] M. E. Madden and M. P. Sarras, “Morphological and biochem-
ical characterization of a human pancreatic ductal cell line
(PANC-1),” Pancreas, vol. 3, no. 5, pp. 512–528, 1988.
[51] S. Ishigaki, N. Hishikawa, J. I. Niwa et al., “Physical and
functional interaction between dorfin and valosin-containing
protein that are colocalized in ubiquitylated inclusions in neu-
rodegenerative disorders,” The Journal of Biological Chemistry,
vol. 279, no. 49, pp. 51376–51385, 2004.
[52] K. E. Hung, V. Faca, K. Song et al., “Comprehensive proteome
analysis of an Apc mouse model uncovers proteins associated
with intestinal tumorigenesis,” Cancer Prevention Research,
vol. 2, no. 3, pp. 224–233, 2009.
[53] J. C. Ghosh, T. Dohi, H. K. Byoung, and D. C. Altieri, “Hsp60
regulation of tumor cell apoptosis,” The Journal of Biological
Chemistry, vol. 283, no. 8, pp. 5188–5194, 2008.
Copyright of Journal of Biomedicine & Biotechnology is the property of Hindawi Publishing Corporation and
its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's
express written permission. However, users may print, download, or email articles for individual use.