Open Access

Membrane proteomic analysis of pancreatic cancer cells

Journal of Biomedical Science201017:74

DOI: 10.1186/1423-0127-17-74

Received: 5 May 2010

Accepted: 13 September 2010

Published: 13 September 2010

Abstract

Background

Pancreatic cancer is one of the most aggressive human tumors due to its high potential of local invasion and metastasis. The aim of this study was to characterize the membrane proteomes of pancreatic ductal adenocarcinoma (PDAC) cells of primary and metastatic origins, and to identify potential target proteins related to metastasis of pancreatic cancer.

Methods

Membrane/membrane-associated proteins were isolated from AsPC-1 and BxPC-3 cells and identified with a proteomic approach based on SDS-PAGE, in-gel tryptic digestion and liquid chromatography with tandem mass spectrometry (LC-MS/MS). X! Tandem was used for database searching against the SwissProt human protein database.

Results

We identified 221 & 208 proteins from AsPC-1 and BxPC-3 cells, respectively, most of which are membrane or membrane-associated proteins. A hundred and nine proteins were found in both cell lines while the others were present in either AsPC-1 or BxPC-3 cells. Differentially expressed proteins between two cell lines include modulators of cell adhesion, cell motility or tumor invasion as well as metabolic enzymes involved in glycolysis, tricarboxylic acid cycle, or nucleotide/lipid metabolism.

Conclusion

Membrane proteomes of AsPC-1 (metastatic) and BxPC-3 (primary) cells are remarkably different. The differentially expressed membrane proteins may serve as potential targets for diagnostic and therapeutic interventions.

Introduction

Pancreatic cancer is one of the most aggressive human malignancies. Despite the advances in therapeutic strategies including surgical techniques as well as local and systemic adjuvant therapies, the overall survival in patients with pancreatic cancer remains dismal and has not improved substantially over the past 30 years. Median survival from diagnosis is typically around 3 to 6 months, and the 5-year survival rate is less than 5%. As a result, in 2003, pancreatic cancer surpassed prostate cancer as the 4th leading cause of cancer-related death in the US [1]. The main reason for the failure of current conventional therapy to cure pancreatic cancer and the major cause for cancer-related mortality in general, is the ability of malignant cells to detach from the primary tumor site and to develop metastasis in different regions of the same organ and in distant organs [2, 3]. Pancreatic cancer usually causes no symptoms early on, leading to locally advanced or metastatic disease at time of diagnosis [4]. In this regard, it is important to identify the functional proteins that regulate/promote metastasis in pancreatic cancer. This would facilitate the development of strategies for therapeutic interventions and improved management of cancer patients.

The purpose of this study is to compare the membrane proteins expressed in pancreatic cancer cells of primary and metastatic origins using a proteomics approach. Membrane proteomics can be defined as analysis and characterization of entire complement of membrane proteins present in a cell under a specific biological condition [5, 6]. In fact, membrane proteins account for more than two-thirds of currently known drug targets. Defining membrane proteomes is therefore important for finding potential drug targets. Membrane proteomics can also serve as a promising approach to human cancer biomarker discovery because membrane proteins are known to have implication in cell proliferation, cell adhesion, cell motility and tumor cell invasion [79].

Materials and methods

Cell culture

AsPC-1 and BxPC-3 cell lines were obtained from American Tissue Culture Collection (ATCC, Rockville, MD). These cell lines were initially generated from patients with pancreatic ductal adenocarcinoma (PDAC) [1012]. The cells were maintained at 5% CO2-95% air, 37°C, and with RPMI 1640 (ATCC) containing 10% FBS, 100 μg/ml penicillin G and 100 mg/ml streptomycin. When the confluence reached 80-90%, the cells were harvested and washed with PBS for three times.

Sample preparation

Membrane proteins from AsPC-1 and BxPC-3 cells were isolated with the ProteoExtract Native Membrane Protein Extraction Kit (EMD Chemicals, Gibbstown, NJ). In brief, the cell pellet was washed three times with the Washing Buffer, and then incubated with ice-cold Extract Buffer |at 4°C for 10 min under gentle agitation. After the pellet was centrifuged at 16,000 g for 15 min (4°C), the supernatant was discarded and 1 mL ice-cold Extract Buffer|| was added to the pellet. This membrane protein extraction step was allowed for 30 min at 4°C under gentle agitation. Then the supernatant was collected after centrifugation at 16,000 g for 15 min 4°C.

SDS-PAGE and proteolytic cleavage

Total membrane protein concentration was measured with the 2-D Quant Kit (GE Healthcare, Piscataway, NJ). In total, 20 μg of membrane proteins from each cell line were loaded into a 4-12% NuPAGE Bis-Tris gel (Invitrogen, Carlsbad, CA) for SDS-PAGE separation. The gel was stained with the Simply Blue staining solution (Invitrogen) to visualize the proteins. Each gel was then cut into 15 sections evenly and proteolytic cleavage of proteins in each section was performed with enzyme-grade trypsin (Promega, Madison, WI) as previously described.

Tandem MS and database searching

Liquid chromatography (LC) with tandem MS (LC/MS/MS) of peptides was performed using a NanoLC system (Eksigent Technologies, Dublin, CA) and a LTQ mass spectrometer (Thermo Fisher, Waltham, MA). Aliquots (5 μL) of the peptide digest derived from each gel slice were injected using an autosampler at a flow rate of 3.5 μL/min. The peptides were concentrated and desalted on a C18 IntegraFrit Nano-Precolumn (New Objective, Woburn, MA) for 10 min, then eluted and resolved using a C18 reversed-phase capillary column (New Objective). LC separation was performed at 400 nL/min with the following mobile phases: A, 5% acetonitrile/0.1%formic acid (v/v); B, 95% acetonitrile/0.1% formic acid (v/v). The chosen LC gradient was: from 5% to 15% B in 1 min, from 15% to 100% B in 40 min, and then maintained at 100%B for 15 min.

Database searches were performed using the X! Tandem search engine against the SwissProt protein sequence database. The search criteria were set with a mass accuracy of 0.4 Da and semi-style cleavage by trypsin. Proteins with two unique peptides are considered as positively identified.

Western blot analysis

AsPC-1 and BxPC-3 cells were lysed with a lysis buffer containing 8 M urea, 2 M Thiourea and 4% CHAPS. Cell lysates with a total protein amount of 40 μg were separated with 8-12% NuPAGE gels at 100 V for about 2 hours and then transferred to polyvinylidene difluoride membrane using an iBlot system (Invitrogen, Carlsbad, CA, USA). After saturating with 2% slim milk, the blots were sequentially incubated with primary antibody (1:100 dilution) and horseradish peroxidase-conjugated antimouse IgG secondary antibody (1:1000 dilution, Applied Biological Materials Inc, Richmond, Canada). Anti-annexin A1 was obtained from Abcam (Cambridge, MA, USA) whereas anti-phosphoglycerate kinase 1 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Finally, the bands were visualized by enhanced chemiluminescence detection (Applied Biological Materials).

Results

The purpose of this study was to demonstrate a membrane proteomic analysis of PDAC cells and to identify differentially expressed membrane proteins between primary and metastatic PDAC cells, which may have a potential role in metastasis of pancreatic cancer. Two PDAC cell lines, AsPC-1 and BxPC-3, were used in this study. AsPC-1 is a cell line of metastatic origin from a 62 year-old female Caucasian whereas BxPC-3 is a cell line of primary PDAC from a 61 year-old female Caucasian [1012]. Membrane proteins of AsPC-1 and BxPC-3 cells were isolated and then resolved with SDS-PAGE (Figure 1A). Proteins in each gel slices were proteolytically cleaved and the resulting peptides were analyzed with LC-MS/MS. In total, we identified 221 and 208 membrane or membrane-associated proteins from AsPC-1 and BxPC-3 cells, respectively, based on at least 2 unique peptides. A hundred and nine proteins were present in both cell lines but others were only found in AsPC-1 or in BxPC-3 cells (Figure 1B). All the identified proteins and matched peptides from the two cell lines are summarized in Additional file 1, Tables S1 and S2. Proteins with single matched peptide were not tabulated although previous publications reported identification of membrane proteins based on single unique peptide [13, 14]. The identified proteins were then sorted according to the Gene Ontology Annotation database (Figure 2). A hundred and four proteins were assigned as membrane proteins in AsPC-1 cells whereas 101 proteins were assigned as membrane proteins in BxPC-3 cells. Table 1 lists the "integral to membrane" proteins found in AsPC-1 and BxPC-3 cells. Besides the membrane proteins, the proteomic analysis also identified many membrane-associated proteins, e.g., extracellular matrix (ECM) proteins. To confirm the proteomic finding, we verified the differential levels of Annexin A1 and PGK1 between AsPC-1 and BxPC-3 cells using Western blotting (Figure 3). Annexin A1 was found to be over-expressed in BxPC-3 cells whereas phosphoglycerate kinase 1 was over-expressed in AsPC-1 cells, which agrees to the results obtained by the proteomic approach.
https://static-content.springer.com/image/art%3A10.1186%2F1423-0127-17-74/MediaObjects/12929_2010_Article_188_Fig1_HTML.jpg
Figure 1

Analysis and identification of membrane proteins in AsPC-1 and BxPC-3 cells using a proteomics approach based on SDS-PAGE, in-gel digestion and LC-MS/MS. (A) Membrane proteins were isolated, separated with SDS-PAGE and detected with Simply Blue stain. The gel bands were then excised and digested with trypsin, and the resulting peptides were extracted for LC-MS/MS analysis. (B) 221 and 208 proteins were identified from AsPC-1 and BxPC-3 cells, respectively, with 109 proteins present in both cell lines.

https://static-content.springer.com/image/art%3A10.1186%2F1423-0127-17-74/MediaObjects/12929_2010_Article_188_Fig2_HTML.jpg
Figure 2

Sorting of the identified proteins according to their subcellular localization.

Table 1

Integral to membrane proteins identified in AsPC-1 & BxPC-3 cells

AsPC-1

 

BxPC-3

 

Accession #

Protein name

Accession #

Protein name

1A25_HUMAN

HLA class I histocompatibility antigen, A-25 alpha chain

4F2_HUMAN

4F2 cell-surface antigen heavy chain

4F2_HUMAN

4F2 cell-surface antigen heavy chain

ACSL3_HUMAN

Long-chain-fatty-acid--CoA ligase 3

AAAT_HUMAN

Neutral amino acid transporter B(0)

ACSL4_HUMAN

Long-chain-fatty-acid--CoA ligase 4

ACSL5_HUMAN

Long-chain-fatty-acid--CoA ligase 5

ADT2_HUMAN

ADP/ATP translocase 2

ADT2_HUMAN

ADP/ATP translocase 2

ALK_HUMAN

ALK tyrosine kinase receptor precursor

ANPRC_HUMAN

Atrial natriuretic peptide clearance receptor

APMAP_HUMAN

Adipocyte plasma membrane-associated protein

AOFB_HUMAN

Amine oxidase [flavin-containing] B

AT1A1_HUMAN

Sodium/potassium-transporting ATPase subunit alpha-1

APMAP_HUMAN

Adipocyte plasma membrane-associated protein

CALX_HUMAN

Calnexin

AT1A1_HUMAN

Sodium/potassium-transporting ATPase subunit alpha-1 precursor

CEAM1_HUMAN

Carcinoembryonic antigen-related cell adhesion molecule 1

ATP7B_HUMAN

Copper-transporting ATPase 2

CEAM6_HUMAN

Carcinoembryonic antigen-related cell adhesion molecule 6

CALX_HUMAN

Calnexin

CKAP4_HUMAN

Cytoskeleton-associated protein 4

CEAM1_HUMAN

Carcinoembryonic antigen-related cell adhesion molecule 1

CLCN1_HUMAN

Chloride channel protein

CEAM6_HUMAN

Carcinoembryonic antigen-related cell adhesion molecule 6

CMC2_HUMAN

Calcium-binding mitochondrial carrier protein Aralar2

CMC2_HUMAN

Calcium-binding mitochondrial carrier protein Aralar2

CODA1_HUMAN

Collagen alpha-1(XIII) chain

CY1_HUMAN

Cytochrome c1, heme protein

CSMD2_HUMAN

CUB and sushi domain-containing protein 2

EGFR_HUMAN

Epidermal growth factor receptor precursor

EAA1_HUMAN

Excitatory amino acid transporter 1

FLNB_HUMAN

Filamin-B

GP124_HUMAN

Probable G-protein coupled receptor 124

FLRT1_HUMAN

Leucine-rich repeat transmembrane protein FLRT1

GRP78_HUMAN

78 kDa glucose-regulated protein

FZD8_HUMAN

Frizzled-8 precursor

HNRPM_HUMAN

Heterogeneous nuclear ribonucleoprotein M

GRP78_HUMAN

78 kDa glucose-regulated protein

ITAV_HUMAN

Integrin alpha-V

IL4RA_HUMAN

Interleukin-4 receptor alpha chain

KCNQ3_HUMAN

Potassium voltage-gated channel subfamily KQT member 3

IMMT_HUMAN

Mitochondrial inner membrane protein

L2HDH_HUMAN

L-2-hydroxyglutarate dehydrogenase

KCNK3_HUMAN

Potassium channel subfamily K member 3

M2OM_HUMAN

Mitochondrial 2-oxoglutarate/malate carrier protein

KTN1_HUMAN

Kinectin

MUC16_HUMAN

Mucin-16

LAMP1_HUMAN

Lysosome-associated membrane glycoprotein 1

MYOF_HUMAN

Myoferlin

LRC59_HUMAN

Leucine-rich repeat-containing protein 59

OST48_HUMAN

Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit

MTCH2_HUMAN

Mitochondrial carrier homolog 2

PCD16_HUMAN

Protocadherin-16 precursor

MUC16_HUMAN

Mucin-16

PGRC1_HUMAN

Membrane-associated progesterone receptor component 1

MYOF_HUMAN

Myoferlin

PHB_HUMAN

Prohibitin

OST48_HUMAN

Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit

PK1L1_HUMAN

Polycystic kidney disease protein 1-like 1

PHB_HUMAN

Prohibitin

PTPRZ_HUMAN

Receptor-type tyrosine-protein phosphatase zeta

S12A1_HUMAN

Solute carrier family 12 member 1

SSRD_HUMAN

Translocon-associated protein subunit delta precursor

SFXN3_HUMAN

Sideroflexin-3

TFR1_HUMAN

Transferrin receptor protein 1

VAT1_HUMAN

Synaptic vesicle membrane protein VAT-1 homolog

TMEDA_HUMAN

Transmembrane emp24 domain-containing protein 10

VDAC2_HUMAN

Voltage-dependent anion-selective channel protein 2

TOM40_HUMAN

Mitochondrial import receptor subunit TOM40 homolog

VMAT2_HUMAN

Synaptic vesicular amine transporter

  
https://static-content.springer.com/image/art%3A10.1186%2F1423-0127-17-74/MediaObjects/12929_2010_Article_188_Fig3_HTML.jpg
Figure 3

Western blot analysis of Annexin A1 and phosphoglycerate kinase 1 (PGK1) between AsPC-1 and BxPC-3 cells.

Discussion

Metastasis is a highly organ-specific process, which requires multiple steps and interactions between tumor cells and the host. These include detachment of tumor cells from the primary tumor, intravasation into lymph and blood vessels, survival in the circulation, extravasation into target organs, and subsequent proliferation and induction of angiogenesis. Many proteins are critically involved in this process, such as cell-cell adhesion molecules (CAMs), members of the cadherins and, integrins, metalloproteinases (MMPs) and the urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) system. As modulators of metastatic growth, these molecules can affect the local ECM, stimulate cell migration, and promote cell proliferation and tumor cell survivals [15]. Furthermore, hypoxia can drive genomic instability and lead to a more aggressive tumor phenotype [16, 17], which may partially explain the highly metastatic nature of PDAC [18]. Last but not least, angiogenesis plays a critical role in invasion and metastasis in terms of tumor cell dissemination. Based on these new insights in mechanism of tumor invasion and metastasis, novel therapies are currently investigated for therapy of patients with pancreatic cancer [1921]. Nevertheless, proteomic analysis of primary and metastatic PDAC is required to reveal additional functional proteins that regulate or promote tumor metastasis, as detailed in previous studies [2224]. These signature molecules are predictors of metastatic risk and also provide a basis for the development of anti-metastatic therapy.

Our proteomic analysis has revealed a large number of differentially expressed membrane/surface proteins between metastatic and primary PDAC cells, and the validity of such a proteomic approach has been verified by Western blot analysis. In fact, the differential expression of membrane proteins between AsPC-1 and BxPC-3 can be observed from the SDS-PAGE patterns of membrane proteins from the two cell lines (Figure 1). The proteins showing differential levels include cadherins, catenin, integrins, galectins, annexins, collagens and many others, which are known to have roles in tumor cell adhesion or motility. Cadherins are a class of type-1 transmembrane proteins that depend on calcium ions to function. They play important roles in cell adhesion, ensuring that cells are bound together within tissues. Catenins, which are proteins found in complexes with cadherins, also mediate cell adhesion. Our study identified cadherins (protocadherin-16 and protocadherin alpha-12) and alpha-2 catenin in primary tumor cells (BxPC-3) but not in metastatic tumor cells (AsPC-1), suggesting a defect in cell-to-cell adhesion in metastatic AcPC-1 cells.

Integrins are members of a glycoprotein family that form heterodimeric receptors for ECM molecules. These proteins are involved in an adhesive function, and they provide traction for movement in cell motility [25]. In total, there are 18 α-subunits and 8 β-subunits, which are paired to form 24 different integrins through non-covalent bonding. Among these proteins, integrin-β1, α2, α5, and α6 represent major adhesion molecules for the adhesion of pancreatic cancer cells to ECM proteins [26]. In our study, integrin-β1 and integrin-β4 was found in both tumor cell lines while integrin α2 and α5 only identified in BxPC-3 cells. Collagens are major ECM proteins. Cell surface-expressed portion of collagens may serve as ligands for integrins, mediating cell-to-cell adhesion. Twelve members of collagen family were found in the BxPC-3 cells whereas only four members found in AsPC-1 cells.

Conversely, galectin-3 and galectin-4 were found in AsPC-1 but not in BxPC-3 cells. Galectins are carbohydrate-binding proteins and have an extremely high affinity for galactosides on cell surface and extracellular glycoproteins. Galectins, especially galectin-3, are modulators of cancer cell adhesion and invasiveness. Galectin-3 usually exists in cytoplasm, but can be secreted and bound on the cell surface by a variety of glycoconjugate ligands. Once localized to the cell surface, galectin-3 is capable of oligomerization, and the resultant cross-linking of surface glycoproteins into multimolecular complexes on the endothelial cell surface is reported to mediate the adhesion of tumor cells to the vascular endothelium [27]. Lysosome-associated membrane glycoprotein 1 (LAMP1) is a receptor for galectin-3, and was found on the cell surface of highly metastatic tumor cells [28]. Our study revealed LAMP1 in AsPC-1 cells but not in BxPC-3 cells. The cell surface-expressed portion of LAMP1 maybe serve as a ligand for galectin 3, mediating cell-cell adhesion and indirectly tumor spread. FKBP12-rapamycin complex-associated protein (a.k.a., mTOR) was also identified in AsPC-1 cells but not in BxPC-3 cells. mTOR is a downstream serine/threonine protein kinase of the phosphatidylinositol 3-kinase/Akt pathway that regulates cell proliferation, cell motility, cell survival, protein synthesis, and transcription. Rapamycin, a specific inhibitor of mTOR, suppresses lymphangiogenesis and lymphatic metastasis in PDAC cells [29].

The described proteomic approach is reproducible for analysis of membrane proteins in cultured pancreatic cancer cells. We observed consistent SDS-PAGE gel patterns for membrane proteins isolated from cultured AsPC-1 or BxPC-3 cells. To examine the reproducibility of LC-MS/MS for identification of membrane proteins, we repeated LC-MS/MS analysis of the peptides yielded from 3 gel bands. Compared to single LC-MS/MS, which identified 45 proteins in total, the duplicate LC-MS/MS analyses identified 47 proteins (~4% increase). This suggested that the observed difference in membrane protein profiles between the two PDAC cell lines is meaningful. Our adopted approach is valid to identify large membrane proteins, which are usually difficult to analyze with 2-D gel electrophoresis (2-DE) method. In AsPC-1 cells, 35% of the identified proteins have a molecular weight above 70 kDa, whereas 43% of the proteins are larger than 70 kDa in BxPC-3 cells. In addition to the proteins either present in AsPC-1 or in BxPC-3 cells, many other proteins were found in both cell types with a differential number of peptides matched. This may reflect the differential level of a protein between the two cell lines, although further verification is needed. Around 50% of the proteins identified in AsPC-1 and BxPC-3 cells are directly classified as membrane proteins, including a number of integral to membrane proteins and plasma membrane proteins. In addition, many mitochondrial inner membrane proteins were also identified from AsPC-1 (n = 21) and BxPC-3 (n = 13) cells. The mitochondrial inner membrane forms internal compartments known as cristae, which allow greater space for the proteins such as cytochromes to function properly and efficiently. The inner mitochondrial membrane contains mitochondria fusion and fission proteins, ATP synthases, transporter proteins regulating metabolite flux as well as proteins that perform the redox reactions of oxidative phosphorylation, many of which were identified in this study. Among the proteins that are not classified as membrane proteins, many are either membrane-associated proteins (e.g., kinases, G proteins, or enzymes) or proteins associated with other subcellular compartments such as mitochondria, endoplasmic reticulum (ER) or nucleus (e.g., histones, elongation factors, translation initiation factor and transcription factors) (Additional file 1, Table S1). It is commonly assumed that a protein is predominantly localized in a given cellular compartment where it exerts its specific function. However, a same protein may be localized at different cell compartments or travel between different organelles and therefore exert multiple cellular functions [30]. In fact, many proteins identified in mitochondria or ER are membrane or membrane-associated proteins.

In addition, many metabolic enzymes were identified from the two PDAC cell lines, reflecting the functional role of pancreas (Tables 2 and 3). These metabolic enzymes are involved in glycolysis, tricarboxylic acid cycle, gluconeogenesis, metabolism of nucleotides, lipids/fatty acids and amino acids, protein folding/unfolded protein response, and pantose phosphate shunt. Table 4 lists the small, membrane associated G proteins identified in AsPC-1 and BxPC-3 cells. Small GTPases regulate a wide variety of cellular processes, including growth, cellular differentiation, cell movement and lipid vesicle transport. RhoA, Rab-1A and Rab-10 were present in AsPC-1 cells whereas Rab-14 was found in BxPC-3 cells. As a proto-oncogene, RhoA regulates a signal transduction pathway linking plasma membrane receptors to the assembly of focal adhesions and actin stress fibers. On the other hand, Rab-1A regulates the 'ER-to-Golgi' transport, a bidirectional membrane traffic between the ER and Golgi apparatus which mediates the transfer of proteins by means of small vesicles or tubular-saccular extensions. Rab-10 is also involved in vesicular trafficking, particularly the directed movement of substances from the Golgi to early sorting endosomes. Mutated KRAS is a potent oncogene in PDAC. KRAS protein is usually tethered to cell membranes because of the presence of an isoprenyl group on its C-terminus. However, KRAS protein was not identified in this study, which might result from numerous mutations of the gene, hindering the matching of peptides based on molecular weight.
Table 2

Metabolic enzymes identified in AsPC-1 cells

Protein name

Accession #

Unique peptides

Total peptides

Mr (Kda)

PI

Biological process

2-oxoglutarate dehydrogenase E1 component, mitochondrial precursor

ODO1_HUMAN

8

18

115.9

6.39

Glycolysis

3,2-trans-enoyl-CoA isomerase, mitochondrial precursor

D3D2_HUMAN

3

13

32.8

8.8

Fatty acid metabolism; Lipid metabolism

3-hydroxyacyl-CoA dehydrogenase type-2

HCD2_HUMAN

6

10

26.9

7.65

Lipid metabolic process; tRNA processing

3-hydroxyisobutyrate dehydrogenase, mitochondrial precursor

3HIDH_HUMAN

7

16

35.3

8.38

Pentose-phosphate shunt; valine metabolic process

3-ketoacyl-CoA thiolase, peroxisomal precursor

THIK_HUMAN

3

4

44.3

8.76

Fatty acid metabolism; Lipid metabolism

3-mercaptopyruvate sulfurtransferase

THTM_HUMAN

3

7

33.2

6.13

Cyanate catabolic process

78 kDa glucose-regulated protein

GRP78_HUMAN

7

12

72.3

5.07

ER-associated protein catabolic process; ER unfolded protein response; ER regulation of protein folding

Acetyl-CoA acetyltransferase, mitochondrial precursor

THIL_HUMAN

2

6

45.2

8.98

Ketone body metabolism

Aconitate hydratase, mitochondrial

ACON_HUMAN

2

3

85.4

7.36

Tricarboxylic acid cycle

Acyl-protein thioesterase 1

LYPA1_HUMAN

2

2

24.7

6.29

Fatty acid metabolism; Lipid metabolism

Adenylate kinase 2, mitochondrial

KAD2_HUMAN

7

20

26.5

7.67

Nucleic acid metabolic process

ADP/ATP translocase 2

ADT2_HUMAN

5

11

32.9

9.76

Transmembrane transporter activity

Aldehyde dehydrogenase, mitochondrial

ALDH2_HUMAN

3

7

56.3

6.63

Alcohol metabolic process

Alpha-enolase

ENOA_HUMAN

2

2

47.1

7.01

Glycolysis

Amine oxidase B

AOFB_HUMAN

2

2

58.7

7.2

Oxidation reduction

Aspartate aminotransferase, mitochondrial

AATM_HUMAN

4

6

47.4

9.14

Lipid transport

ATP synthase subunit alpha, mitochondrial

ATPA_HUMAN

21

52

59.7

9.16

ATP synthesis

ATP synthase subunit d, mitochondrial

ATP5H_HUMAN

3

7

18.5

5.21

ATP synthesis; Ion transport

ATP synthase subunit b, mitochondrial

AT5F1_HUMAN

2

3

28.9

9.37

ATP synthesis

ATP synthase subunit beta, mitochondrial

ATPB_HUMAN

28

95

56.5

5.26

ATP synthesis

ATP synthase subunit f, mitochondrial

ATPK_HUMAN

2

2

10.9

9.7

ATP synthesis; Ion transport

ATP synthase subunit gamma, mitochondrial;

ATPG_HUMAN

3

6

33

9.23

ATP synthesis; proton transport

ATP synthase subunit O, mitochondrial

ATPO_HUMAN

6

11

23.3

9.97

ATP synthesis, ion transport; ATP catabolic process

Calcium-binding mitochondrial carrier protein Aralar2

CMC2_HUMAN

7

16

74.1

7.14

Mitochondrial aspartate and glutamate carrier

Citrate synthase, mitochondrial precursor

CISY_HUMAN

2

3

51.7

8.45

Tricarboxylic acid cycle

Cytochrome b5 type B

CYB5B_HUMAN

2

4

16.3

4.88

Electron transport

Cytochrome b-c1 complex subunit 1, mitochondrial

QCR1_HUMAN

6

12

52.6

5.94

Electron transport

Cytochrome b-c1 complex subunit 2, mitochondrial

QCR2_HUMAN

3

4

48.4

8.74

Aerobic respiration; electron transport chain; oxidative phosphorylation

Cytochrome c oxidase subunit 2

COX2_HUMAN

2

6

25.5

4.67

Electron transport chain

Cytochrome c1, heme protein, mitochondrial

CY1_HUMAN

5

10

35.4

9.15

Electron transport chain

Cytochrome c1, heme protein, mitochondrial

CY1_HUMAN

2

3

35.4

9.15

Electron transport chain

D-beta-hydroxybutyrate dehydrogenase, mitochondrial precursor

BDH_HUMAN

2

3

38.1

9.1

Oxidation reduction

Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, mitochondrial

ECH1_HUMAN

4

10

35.8

8.16

Fatty acid metabolism; Lipid metabolism

Delta-1-pyrroline-5-carboxylate synthetase

P5CS_HUMAN

2

4

87.2

6.66

Amino-acid biosynthesis; Proline biosynthesis

Dihydrolipoyl dehydrogenase, mitochondrial

DLDH_HUMAN

7

16

54.1

7.95

Cell redox homeostasis

Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial

ODP2_HUMAN

3

5

65.7

7.96

Glycolysis

Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial

ODO2_HUMAN

4

7

48.6

9.01

Tricarboxylic acid cycle

Electron transfer flavoprotein subunit alpha, mitochondrial

ETFA_HUMAN

2

5

35.1

8.62

Electron transport

Electron transfer flavoprotein subunit beta

ETFB_HUMAN

4

6

27.8

8.25

Electron transport

Endoplasmin

ENPL_HUMAN

16

28

92.4

4.76

ER-associated protein catabolic process; protein folding/transport; response to hypoxia

Enoyl-CoA hydratase, mitochondrial

ECHM_HUMAN

9

26

31.4

8.34

Fatty acid metabolism; Lipid metabolism

Glutamate dehydrogenase 1, mitochondrial;

DHE3_HUMAN

3

4

61.4

7.66

Glutamate metabolism

Glyceraldehyde-3-phosphate dehydrogenase

G3P_HUMAN

5

7

36

8.57

Glycolysis

Glycerol-3-phosphate dehydrogenase, mitochondrial precursor

GPDM_HUMAN

8

15

80.8

7.23

Glycolysis

Haloacid dehalogenase-like hydrolase domain-containing protein 3

HDHD3_HUMAN

3

4

28

6.21

Metabolic process phosphoglycolate phosphatase activity

Histidine triad nucleotide-binding protein 2

HINT2_HUMAN

2

3

17.2

9.2

Lipid synthesis; Steroid biosynthesis

Hyaluronidase-3

HYAL3_HUMAN

2

2

46.5

 

Carbohydrate metabolic process

Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial precursor

HCDH_HUMAN

2

4

34.3

8.88

Fatty acid metabolism; Lipid metabolism

Isoleucyl-tRNA synthetase, mitochondrial precursor

SYIM_HUMAN

5

7

113.7

6.78

Protein biosynthesis

Isovaleryl-CoA dehydrogenase, mitochondrial

IVD_HUMAN|

2

2

46.3

8.45

Leucine catabolic process; Oxidation reduction

L-lactate dehydrogenase A chain

LDHA_HUMAN

3

5

36.7

8.84

Glycolysis

Lon protease homolog, mitochondrial

LONM_HUMAN

2

2

106.4

6.01

Required for intramitochondrial proteolysis

Long-chain-fatty-acid--CoA ligase 5;

ACSL5_HUMAN

2

4

75.9

6.49

Fatty acid metabolism; Lipid metabolism

Malate dehydrogenase, mitochondrial

MDHM_HUMAN

3

5

35.5

8.92

Tricarboxylic acid cycle; Glycolysis

Medium-chain specific acyl-CoA dehydrogenase, mitochondrial

ACADM_HUMAN

2

6

46.6

8.61

Fatty acid metabolism; Lipid metabolism

Mitochondrial carrier homolog 2

MTCH2_HUMAN

3

10

33.3

8.25

Transmembrane transport

Mitochondrial inner membrane protein

IMMT_HUMAN

2

2

83.6

6.08

Protein binding; Cell proliferation-inducing

NADH-cytochrome b5 reductase 3

NB5R3_HUMAN

3

3

34.2

7.18

Cholesterol biosynthesis; Lipid/steroid synthesis

Neutral alpha-glucosidase AB

GANAB_HUMAN

6

9

106.8

5.74

Carbohydrate metabolic process

Peptidyl-prolyl cis-trans isomerase A

PPIA_HUMAN

2

3

18

7.68

Protein folidng; Interspecies interation

Peroxiredoxin-5

PRDX5_HUMAN

2

5

22

8.85

Cell redox homeostasis

Phosphoenolpyruvate carboxykinase, mitochondrial

PPCKM_HUMAN

8

18

70.6

7.56

Gluconeogenesis

Phosphoglycerate kinase 1

PGK1_HUMAN

4

7

44.6

8.3

Glycolysis

Protein disulfide-isomerase

PDIA1_HUMAN

3

3

57.1

4.76

Cell redox homeostasis

Protein disulfide-isomerase A3

PDIA3_HUMAN

4

7

56.7

5.98

Cell redox homeostasis

Protein disulfide-isomerase A4

PDIA4_HUMAN

2

2

72.9

4.96

Cell redox homeostasis; Protein secretion

Protein disulfide-isomerase A6

PDIA6_HUMAN

2

3

48.1

4.95

Cell redox homeostasis; Protein folding

Protein ETHE1, mitochondrial

ETHE1_HUMAN

4

11

27.9

6.35

Metabolic homeostasis in mitochondria

Protein transport protein Sec16A

SC16A_HUMAN

2

2

233.4

5.4

ER-Golgi transport; Protein transport

Pyruvate dehydrogenase E1 component alpha subunit, somatic form

ODPA_HUMAN

2

4

43.3

8.35

Glycolysis

Pyruvate dehydrogenase E1 component subunit alpha, mitochondrial precursor

ODPAT_HUMAN

3

7

42.9

8.76

Glycolysis

Pyruvate dehydrogenase E1 component subunit beta, mitochondrial

ODPB_HUMAN

2

3

39.2

6.2

Glycolysis; Tricarboxylic acid cycle

Serine hydroxymethyltransferase, mitochondrial

GLYM_HUMAN

12

21

56

8.76

L-serine metabolic process; Glycine metabolic process; One-carbon metabolic process

Succinate dehydrogenase flavoprotein subunit, mitochondrial

DHSA_HUMAN

2

5

72.6

7.06

Electron transport; Tricarboxylic acid cycle

Succinyl-CoA ligase [GDP-forming] beta-chain, mitochondrial precursor

SUCB2_HUMAN

3

3

46.5

6.15

Succinyl-CoA metabolic process; Tricarboxylic acid cycle

Succinyl-CoA ligase [GDP-forming] subunit alpha, mitochondrial precursor

SUCA_HUMAN

2

5

35

9.01

Tricarboxylic acid cycle

Superoxide dismutase [Mn], mitochondrial

SODM_HUMAN

2

5

24.7

8.35

Elimination of radicals

Thioredoxin-dependent peroxide reductase

PRDX3_HUMAN

4

10

27.7

7.68

Cell redox homeostasis; Hydrogen peroxide catabolic process

Thiosulfate sulfurtransferase

THTR_HUMAN

2

3

33.4

6.77

Cyanate catabolic process

Trifunctional enzyme subunit alpha, mitochondrial

ECHA_HUMAN

17

46

82.9

9.16

Fatty acid metabolism; Lipid metabolism

Trifunctional enzyme subunit beta, mitochondrial

ECHB_HUMAN

6

12

51.3

9.45

Fatty acid metabolism

Trimethyllysine dioxygenase, mitochondrial

TMLH_HUMAN

2

3

49.5

7.64

Carnitine biosynthesis

Very long-chain specific acyl-CoA dehydrogenase, mitochondrial

ACADV_HUMAN

3

5

70.3

8.92

Fatty acid metabolism; Lipid metabolism

Table 3

Metabolic enzymes identified in BxPC-3 cells

Protein name

Accession #

Unique peptides

Total peptides

Mr (KDa)

PI

Biological process

2-oxoglutarate dehydrogenase E1 component, mitochondrial

ODO1_HUMAN

4

4

115.9

6.39

Glycolysis

3-ketoacyl-CoA thiolase, mitochondrial

THIM_HUMAN

2

4

41.9

8.32

Fatty acid metabolism Lipid metabolism

78 kDa glucose-regulated protein

GRP78_HUMAN

31

91

72.3

5.07

ER-associated protein catabolic process ER unfolded protein response ER regulation of protein folding

Adenylate kinase 2, mitochondrial

KAD2_HUMAN

4

7

26.5

7.67

Nucleotide/nucleic acid metabolic process

ADP/ATP translocase 2

ADT2_HUMAN

2

5

32.9

9.76

Transmembrane transporter activity

Alpha-aminoadipic semialdehyde dehydrogenase

AL7A1_HUMAN

2

2

55.3

6.44

Cellular aldehyde metabolic process; oxidation reduction

Alpha-enolase

ENOA_HUMAN

3

5

47.1

7.01

Glycolysis

Annexin A1

ANXA1_HUMAN

4

5

38.7

6.57

Anti-apoptosis; Exocytosis; Lipid metabolic process

Aspartate aminotransferase, mitochondrial precursor

AATM_HUMAN

2

7

47.4

9.14

Lipid transport

ATP synthase subunit alpha, mitochondrial

ATPA_HUMAN

3

6

59.7

9.16

ATP synthesis

ATP synthase subunit beta, mitochondrial

ATPB_HUMAN

4

13

56.5

5.26

ATP synthesis

ATP synthase subunit d, mitochondrial

ATP5H_HUMAN

2

4

18.5

5.21

ATP synthesis; Ion transport

ATP synthase subunit gamma, mitochondrial

ATPG_HUMAN

2

3

33

9.23

ATP synthesis; Proton transport

ATP synthase subunit O, mitochondrial

ATPO_HUMAN

2

3

23.3

9.97

ATP synthesis; Ion transport ATP catabolic process

Calcium-binding mitochondrial carrier protein Aralar2

CMC2_HUMAN

2

4

74.1

7.14

Mitochondrial aspartate and glutamate carrier

Citrate synthase, mitochondrial;

CISY_HUMAN

3

5

51.7

8.45

Tricarboxylic acid cycle

Cytochrome b-c1 complex subunit 1, mitochondrial

QCR1_HUMAN

3

5

52.6

5.94

Electron transport

Cytochrome b-c1 complex subunit 2, mitochondrial

QCR2_HUMAN

2

2

48.4

8.74

Aerobic respiration; Electron transport chain; Oxidative phosphorylation

Cytochrome c oxidase subunit 2

COX2_HUMAN

2

4

25.5

4.67

Electron transport chain

Cytochrome c oxidase subunit 5B, mitochondrial precursor

COX5B_HUMAN

2

2

13.7

9.07

Respiratory gaseous exchange

Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, mitochondrial precursor

ECH1_HUMAN

2

6

35.8

8.16

Fatty acid metabolism; Lipid metabolism

Delta-1-pyrroline-5-carboxylate synthetase

P5CS_HUMAN

2

3

87.2

6.66

Amino-acid biosynthesis; Proline biosynthesis

Dihydrolipoyl dehydrogenase, mitochondrial

DLDH_HUMAN

5

13

54.1

7.95

Cell redox homeostasis

Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial

ODO2_HUMAN

3

6

48.6

9.01

Tricarboxylic acid cycle

Electron transfer flavoprotein subunit alpha, mitochondrial

ETFA_HUMAN

3

7

35.1

8.62

Electron transport

Electron transfer flavoprotein subunit beta

ETFB_HUMAN

2

3

27.8

8.25

Electron transport

Endoplasmin

ENPL_HUMAN

16

31

92.4

4.76

ER-associated protein catabolic process; protein folding/transport; response to hypoxia

Enoyl-CoA hydratase, mitochondrial

ECHM_HUMAN

3

12

31.4

8.34

Fatty acid metabolism; Lipid metabolism

ERO1-like protein alpha precursor

ERO1A_HUMAN

2

3

54.4

5.48

Electron transport

Glucosidase 2 subunit beta

GLU2B_HUMAN

2

5

59.4

4.33

ER protein kinase cascade

Glutamate dehydrogenase 1, mitochondrial

DHE3_HUMAN

2

2

61.4

7.66

Glutamate metabolism

Glyceraldehyde-3-phosphate dehydrogenase

G3P_HUMAN

2

2

36

8.57

Glycolysis

Glycerol-3-phosphate dehydrogenase, mitochondrial

GPDM_HUMAN

2

4

80.8

7.23

Glycolysis

Heme oxygenase 2

HMOX2_HUMAN

2

4

36

5.31

Heme oxidation; Oxidation reduction; Response to hypoxia

Hexokinase-1

HXK1_HUMAN

2

3

102.4

6.36

Glycolysis

L-2-hydroxyglutarate dehydrogenase, mitochondrial

L2HDH_HUMAN

2

2

50.3

8.57

Cellular protein metabolic process; Oxidation reduction

Lon protease homolog, mitochondrial

LONM_HUMAN

2

2

106.4

6.01

Required for intramitochondrial proteolysis

Long-chain-fatty-acid--CoA ligase 3

ACSL3_HUMAN

2

3

80.4

8.65

Fatty acid metabolism; Lipid metabolism

Long-chain-fatty-acid--CoA ligase 4

ACSL4_HUMAN

2

3

79.1

8.66

Fatty acid metabolism; Lipid metabolism

Malate dehydrogenase, mitochondrial

MDHM_HUMAN

3

4

35.5

8.92

TCA glycolysis

Medium-chain specific acyl-CoA dehydrogenase, mitochondrial

ACADM_HUMAN|

2

3

46.6

8.61

Fatty acid metabolism; Lipid metabolism

Methylenetetrahydrofolate reductase

MTHR_HUMAN

2

2

74.5

5.22

Methionine metabolic process; Oxidation reduction

Mitochondrial 2-oxoglutarate/malate carrier protein

M2OM_HUMAN

2

2

34

9.92

Transport

Mitochondrial import receptor subunit TOM40 homolog

TOM40_HUMAN

3

3

37.9

6.79

Ion transport; Protein transport

Neutral alpha-glucosidase AB

GANAB_HUMAN

7

10

106.8

5.74

Carbohydrate metabolic process

Neutral cholesterol ester hydrolase 1

ADCL1_HUMAN

2

4

45.8

6.76

Lipid degradation

Ornithine aminotransferase, mitochondrial precursor

OAT_HUMAN

4

6

48.5

6.57

Mitochondrial matrix protein binding

Phosphoenolpyruvate carboxykinase, mitochondrial

PPCKM_HUMAN

2

3

70.6

7.56

Gluconeogenesis

Protein disulfide-isomerase

PDIA1_HUMAN

8

14

57.1

4.76

Cell redox homeostasis

Protein disulfide-isomerase A3

PDIA3_HUMAN

16

25

56.7

5.98

Cell redox homeostasis

Protein disulfide-isomerase A4

PDIA4_HUMAN

7

11

72.9

4.96

Cell redox homeostasis; Protein secretion

Protein disulfide-isomerase A6

PDIA6_HUMAN

2

4

48.1

4.95

Cell redox homeostasis; Protein folding

Pyruvate kinase isozymes M1/M2

KPYM_HUMAN

5

7

57.9

7.96

Glycolysis; Programmed cell death

Serine hydroxymethyltransferase, mitochondrial precursor

GLYM_HUMAN

2

4

56

8.76

L-serine metabolic process; Glycine metabolic process; One-carbon metabolic process

Sterol regulatory element-binding protein 2

SRBP2_HUMAN

2

2

123.6

8.72

Cholesterol metabolism; Lipid metabolism; Steroid metabolism;

Succinate dehydrogenase flavoprotein subunit, mitochondrial

DHSA_HUMAN

3

10

72.6

7.06

Electron transport; Tricarboxylic acid cycle

Succinyl-CoA:3-ketoacid-coenzyme A transferase 1

SCOT_HUMAN

2

5

56.1

7.13

Ketone body catabolic process

Sulfide:quinone oxidoreductase, mitochondrial

SQRD_HUMAN

6

9

49.9

9.18

Oxidation reduction

Superoxide dismutase [Mn], mitochondrial

SODM_HUMAN

2

5

24.7

8.35

Elimination of radicals

Transmembrane emp24 domain-containing protein 10

TMEDA_HUMAN

2

3

25

6.98

ER-Golgi protein transport

Trifunctional enzyme subunit alpha, mitochondrial

ECHA_HUMAN

4

7

82.9

9.16

Fatty acid metabolism; Lipid metabolism

Trifunctional enzyme subunit beta, mitochondrial

ECHB_HUMAN

2

4

51.3

9.45

Fatty acid metabolism

Table 4

A list of small G proteins identified in AsPC-1 and BxPC-3 cells

AsPC-1

      

Ras-related protein Rab-1B

3

7

22.2

 

RAB1B_HUMAN

VVDNTTAKEF ADSLGIPFLE TSAK

      

VVDNTTAKEF ADSLGIPFLE TSAK

      

EFADSLGIPF LETSAK

      

EFADSLGIPF LETSAK

      

EFADSLGIPF LETSAK

      

EFADSLGIPF LETSAK

      

NATNVEQAFM TMAAEIK

Ras-related protein Rab-7a

3

5

23.5

 

RAB7A_HUMAN

DPENFPFVVL GNKIDLENR

      

DPENFPFVVL GNKIDLENR

      

DPENFPFVVL GNK

      

EAINVEQAFQ TIAR

      

EAINVEQAFQ TIAR

Ras-related protein Rab-1A

3

7

22.7

 

RAB1A_HUMAN

VVDYTTAKEF ADSLGIPFLE TSAK

      

VVDYTTAKEF ADSLGIPFLE TSAK

      

EFADSLGIPF LETSAK

      

EFADSLGIPF LETSAK

      

EFADSLGIPF LETSAK

      

EFADSLGIPF LETSAK

      

NATNVEQSFM TMAAEIK

Ras-related protein Rab-10;

2

6

22.5

8.58

RAB10_HUMAN

LLLIGDSGVG K

      

LLLIGDSGVG K

      

AFLTLAEDIL R

      

AFLTLAEDIL R

      

AFLTLAEDIL R

      

AFLTLAEDIL R

Ras-related protein Rab-2A

3

3

23.5

6.08

RAB2A_HUMAN

YIIIGDTGVG K

      

TASNVEEAFI NTAK

      

IGPQHAATNA THAGNQGGQQ AGGGCC

Ras GTPase-activating-like protein IQGAP1

2

2

189.1

 

IQGA1_HUMAN

ILAIGLINEA LDEGDAQK

      

FQPGETLTEI LETPATSEQE AEHQR

Transforming protein RhoA

2

3

21.8

 

RHOA_HUMAN

QVELALWDTA GQEDYDR

      

QVELALWDTA GQEDYDR

      

HFCPNVPIIL VGNKK

BxPC-3

      

Ras-related protein Rab-2A

2

3

23.5

6.08

RAB2A_HUMAN

GAAGALLVYD ITR

      

TASNVEEAFI NTAK

      

TASNVEEAFI NTAK

Ras-related protein Rab-1B

3

8

22.2

5.55

RAB1B_HUMAN

VVDNTTAKEF ADSLGIPFLE TSAK

      

VVDNTTAKEF ADSLGIPFLE TSAK

      

VVDNTTAKEF ADSLGIPFLE TSAK

      

EFADSLGIPF LETSAK

      

EFADSLGIPF LETSAK

      

EFADSLGIPF LETSAK

      

EFADSLGIPF LETSAK

      

NATNVEQAFM TMAAEIK

Ras-related protein Rab-7a

2

3

23.5

6.39

RAB7A_HUMAN

DPENFPFVVL GNK

      

EAINVEQAFQ TIAR

      

EAINVEQAFQ TIAR

Ras-related protein Rab-14

2

2

23.9

5.85

RAB14_HUMAN

TGENVEDAFL EAAKK

      

TGENVEDAFL EAAK

Cell division control protein 42 homolog

2

3

21.3

5.76

CDC42_HUMAN

TPFLLVGTQI DLRDDPSTIE K

      

TPFLLVGTQI DLRDDPSTIE K

      

TPFLLVGTQI DLR

Guanine nucleotide-binding protein subunit beta-2

2

4

37.3

5.6

GBB2_HUMAN

SELEQLRQEA EQLR

      

SELEQLRQEA EQLR

      

KACGDSTLTQ ITAGLDPVGR

      

KACGDSTLTQ ITAGLDPVGR

Some of the proteins identified from the current study may be further verified in clinical specimens as biomarkers for diagnostic/prognostic applications. Particularly, protein biomarkers may be used to classify pancreatic cancer patients for a better treatment decision. Cancer biomarker discovery is an intensive research area. Despite the fact that a large number of researchers are searching for cancer biomarkers, only a handful of protein biomarkers have been approved by the US Food and Drug Administration (FDA) for clinical use [31]. Interestingly, most of the FDA-approved protein biomarkers for human cancers are membrane proteins, including cancer antigen CA125 (ovarian), carcinoembryonic antigen (colon), epidermal growth factor receptor (colon), tyrosine-protein kinase KIT (gastrointestinal), HER2/NEU, CA15-3, CA27-29, Oestrogen receptor and progesterone receptor (breast) and bladder tumour-associated antigen (bladder) [31]. Similarly, most of the reported protein biomarkers in PDAC are of membrane origin or membrane-associated, including CA 19-9, CEA, CA 242, CA 72-4, KRAS, KAI1, CEA-related cell adhesion molecule 1 (CEACAM1), MUC1, MUC4, among many others [3239]. For instance, CA 19-9 is a membrane carbohydrate antigen and the most commonly used biomarker in pancreatic cancers. As a cell adhesion molecule, CEA actually mediates the collagen binding of epithelial cells [40]. KAI1, a metastasis suppressor protein, belongs to the transmembrane 4 superfamily. It is up-regulated in early PDAC and down-regulated in metastatic PDAC [34]. The present study also identified CEA-related cell adhesion molecule 1, CEA-related cell adhesion molecule 6, 4F2 cell-surface antigen heavy chain (a.k.a., CD98), epidermal growth factor receptor (EGFR), hypoxia up-regulated protein 1, MUC16 and mTOR, which may be further verified in clinical specimens as biomarkers for PDAC.

In summary, we have demonstrated a proteomic approach for analysis and identification of membrane proteins in primary and metastatic PDAC cells. Many of the identified proteins are known to be modulators of cell-to-cell adhesion and tumor cell invasion. With the potential targets derived from the present study, we will next focus on promising candidates and explore their functional role in cell proliferation, apoptosis or metabolism in PDAC. Similar membrane proteomics approach can be applied to tissue specimens from patients with primary and metastatic tumors to reveal membrane protein targets for prognostic application or therapeutic intervention.

Declarations

Authors’ Affiliations

(1)
UCLA School of Dentistry & Dental Research Institute
(2)
UCLA Center of Excellence in Pancreatic Diseases
(3)
UCLA Jonsson Comprehensive Cancer Center

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