Membrane proteomic analysis of pancreatic cancer cells

  • Xiaojun Liu1,

    Affiliated with

    • Min Zhang1,

      Affiliated with

      • Vay Liang W Go2 and

        Affiliated with

        • Shen Hu1, 3Email author

          Affiliated with

          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.
          http://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.

          http://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

            
          http://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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