Proteomic Analysis of Pancreatic Ductal Adenocarcinoma Cells

ARTICLE pubs.acs.org/jpr

Proteomic Analysis of Pancreatic Ductal Adenocarcinoma Cells Reveals Metabolic Alterations Weidong Zhou,*,‡ Michela Capello,§,^ Claudia Fredolini,‡,§,^ Lorenzo Piemonti,† Lance A. Liotta,‡ Francesco Novelli,§,^ and Emanuel F. Petricoin‡ ‡

Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, Virginia 20110, United States Center for Experimental Research and Medical Studies, San Giovanni Battista Hospital, Turin 10126, Italy ^ Department of Medicine and Experimental Oncology, University of Turin, Turin 10125, Italy † Diabetes Research Institute, San Raffaele Scientific Institute, Milano 20132, Italy §

bS Supporting Information ABSTRACT: In this present work, we characterized the proteomes of pancreatic ductal adenocarcinoma (PDAC) cells and normal pancreatic duct cells by mass spectrometry using LTQOrbitrap, and identified more than 1200 proteins from each sample. On the basis of spectra count label-free quantification approach, we identified a large number of differentially expressed metabolic enzymes and proteins involved in cytoskeleton, cell adhesion, transport, transcription, translation, and cell proliferation as well. The data demonstrated that metabolic pathways were altered in PDAC, consistent with Warburg effect. Our analysis provides a potentially comprehensive picture of metabolism in PDAC, which may serve as the basis of new diagnostic and treatment of PDAC. KEYWORDS: mass spectrometry, LTQ-Orbitrap, proteomics, pancreatic ductal adenocarcinoma, metabolism, Warburg effect

1. INTRODUCTION Today, liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) is used routinely for large-scale protein identification and global profiling of post-translational modifications from complex biological mixtures.1-4 To quantitatively characterize and compare two or more proteomes by MS, a variety of methods involving the incorporation of stable isotope labels have been developed.5 Because the stable isotope-labeled peptides possess similar physical and chemical properties as their unlabeled equivalents but with a different mass that can be recognized by a mass spectrometer, quantification is achieved by comparing their respective signal intensities. The labels can be introduced into samples by methods such as isotope coded affinity tag (ICAT),6 isotope tags for relative and absolute quantification (iTRAQ),7 and stable isotope labeling by amino acids in cell culture (SILAC).8 However, in many cases, clinical samples and most animal based samples may not be capable of metabolic labeling, and chemical isotopic labeling of many samples can be extremely expensive and time prohibitive.9 As a result, alternative label-free quantification approaches, either by measuring and comparing the MS signal intensity of peptide precursor ions or by counting and comparing the number of matched MS2 spectra of a given protein, have gained increasing popularity over the past several years as investigators r 2011 American Chemical Society

have validated this approach as a viable method for MS-based differential display and shown excellent correlation between spectral counts and relative quantitation.10-12 Pancreatic cancer is the fourth leading cause of cancer death in the United States and Europe. The absence of early symptoms or clinical-pathological markers results in diagnosis at a late, inoperable stage in more than 80% of cases. Most patients die within 12 months and only 4% survive for 5 years after diagnosis.13,14 In the past decade, various MS-based quantitative approaches have been applied to investigate the proteomes of diseased and normal samples from pancreatic tissues, juice, cell lines, and serum, with the goals of dissecting the abnormal signaling pathways underlying oncogenesis and identifying new biomarkers.15-24 These studies identified dozens of differentially expressed proteins, involving in tumor growth, migration, angiogenesis, invasion, metastasis, and immunologic escape. In an effort to systematically reveal protein expression in pancreatic cancer, particularly focusing on the metabolism, we performed proteomic analysis of pancreatic ductal adenocarcinoma (PDAC) cells and normal pancreatic duct cells by LC-MS/MS using an LTQ-Orbitrap mass spectrometer, and identified a large number of differentially expressed metabolic Received: November 24, 2010 Published: February 10, 2011 1944

dx.doi.org/10.1021/pr101179t | J. Proteome Res. 2011, 10, 1944–1952

Journal of Proteome Research

ARTICLE

Figure 1. Chromatogram of LC-MS/MS analysis of tryptic peptides from normal pancreatic duct and CFPAC-1 cells. Twenty micrograms of tryptic peptides and 100 fmol standard peptide AngI were loaded to C18 capillary column, and the eluted peptides were analyzed by LTQ-Orbitrap. For the normal duct sample, the retention time of exogenous control AngI was 78 min, and the retention times of 4 internal control peptides were 30, 41, 51, and 81 min, respectively. Labeled peak A is the peptide IWHHTFYNELR (2þ ion, m/z 758.3768) from actin, gamma 1; peak B is the peptide VAPEEHPVLLTEAPLNPK (2þ ion, m/z 977.5368) from actin, gamma 1; peak C is the peptide LGEHNIDVLEGNEQFINAAK (2þ ion, m/z 1106.0548) from trypsin autolysis; peak D is the peptide LCYVALDFEQEMATAASSSSLEK (2þ ion, m/z 1275.5951) from actin, gamma 1. These peptides were also identified in CFPAC-1 sample with similar retention times ((1.5 min), indicating that the experimental procedure was reproducible and the MS results could be compared.

enzymes based on spectra count label-free quantification approach. The result illuminates the fundamental metabolic alterations in PDAC, concurring with observations from other cancers,25 and provides insight into the biochemical foundation for selective pharmacological probes to perturb these proteins in PDAC.

2. MATERIALS AND METHODS 2.1. Sample Preparation

CFPAC-1 cells (metastatic cell line derived from PDAC patients, ECACC ref no.: 91112501) were cultured at 37 C in Dulbecco modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 20 mM glutamine, 10% fetal calf serum (FCS), and 40 μg/mL Gentamycin with humidified 5% CO2. The cells were harvested and washed with Hank’s balanced salt solution (Sigma-Aldrich). The cell pellet was freeze-dried overnight and stored at -80 C until use. Normal human pancreatic duct cells were obtained from a single brain death donor under IRB approval (San Raffaele Scientific Institute, Italy). The CFPAC-1 cells and normal duct cells were resuspended for 1 h in lysis buffer consisting of Tris/HCl (50 mM, pH 7.4), NaCl (150 mM), Triton X-100 (0.5% w/v), NP-40 (0.5% w/v), 80 mM dithiothreitol (DTT), 10 μL/mL protease inhibitor cocktails (Sigma-Aldrich), 1 mM PMSF, 1 mM Na3VO4 and PhosStop phosphatase inhibitor cocktail (Roche), sonicated for 30 s, and centrifuged at 16 000g for 10 min. The supernatants were precipitated with 4 vol of acetone (Sigma-Aldrich) overnight at -20 C and centrifuged at 9000g for 5 min. The pellets were dried by lyophylization (Heto, Dry Winner) for 2 h. 2.2. Trypsin Digestion and Desalting

The cell pellets were resuspended in 200 μL of 8 M urea, and the protein concentration was measured by Bradford Assay (BioRad). The proteins were transferred to a 1.5-mL eppendorf

tube, reduced by 10 mM dithiothreitol (DTT) for 30 min at 37 C, and then alkylated by 50 mM iodoacetamide for 20 min at room temperature. The concentrated urea in the sample was diluted to a final concentration of 2 M, and the proteins were digested by trypsin at 37 C for 6 h in a buffer containing ammonium bicarbonate (50 mM, pH 9). The digestion mixture was then acidified by adding glacial acetic acid to a final concentration of 2% and desalted by ZipTip (Millipore). 2.3. Mass Spectrometry for Peptide Identification

The peptides were analyzed by high sensitive reversed-phase liquid chromatography coupled nanospray tandem mass spectrometry (LC-MS/MS) using an LTQ-Orbitrap mass spectrometer (Thermo Fisher).26 The reversed-phase LC column was slurrypacked in-house with 5 μm, 200 Å pore size C18 resin (Michrom BioResources, CA) in a 100 μm i.d. 10 cm long piece of fused silica capillary (Polymicro Technologies, Phoenix, AZ) with a laser-pulled tip. After packing, the new column, the HPLC system (Surveyor MS Pump Plus from ThermoFisher) and the LTQOrbitrap were tested by analyzing 100 fmol “Yeast Enolase Standard & Tryptic Digestion” from Michrom Bioresources, Inc. (catalogue number PTD/00001/46) to ensure that stable ESI, desired mass accuracy, peak resolution, peak intensity and retention time could be obtained. Additional iteration was performed to ensure reproducibility. A total of 100 fmol of standard peptide angiotensin I (Ang I) was spiked into the sample as an internal standard. After sample injection, the column was washed for 5 min with mobile phase A (0.1% formic acid), and peptides were eluted using a linear gradient of 0% mobile phase B (0.1% formic acid, 80% acetonitrile) to 50% B in 120 min at 200 nL/min, then to 100% B in an additional 10 min for the proteomics analysis. Before and after analyzing one sample, the column was washed with HPLC mobile phase B for 30 min, then mobile phase A for 20 min at high flow rate (1 μL/min) to reduce potential carryover. 1945

dx.doi.org/10.1021/pr101179t |J. Proteome Res. 2011, 10, 1944–1952


ARTICLE

Table 1. A Partial List of Up-Regulated Metabolic Proteins in PDAC proteins

functional annotationa

accession

spectra count in

spectra count

number

normal duct

in PDAC

phosphofructokinase, muscle

glycolysis

4505749

1

4

pyruvate kinase, muscle isoform M1

glycolysis

33286420

35

63 67

pyruvate kinase, muscle isoform M2

glycolysis

33286418

14

citrate synthase precursor

Krebs cycle

38327625

2

8

L-lactate

pyruvate fermentation

4557032

4

53

dehydrogenase B

glucose-6-phosphate dehydrogenase isoform b

pentose phosphate pathway

108773793

0

33

phosphogluconate dehydrogenase transketolase isoform 1

pentose phosphate pathway pentose phosphate pathway

40068518 4507521

8 22

24 47

ATP citrate lyase isoform 1

fatty acid synthesis

38569421

4

24

fatty acid synthase

fatty acid synthesis

41872631

0

108

acyl-CoA thioesterase 7 isoform hBACHa

fatty acid metabolism

75709208

0

5

acyl-CoA synthetase long-chain family member 3

fatty acid metabolism

42794752

2

7

brain glycogen phosphorylase

glycogen degradation

21361370

11

56

phosphoribosylaminoimidazole succinocarboxamide

purine synthesis

5453539

0

26

synthetase isoform 2 phosphoribosylglycinamide formyltransferase

purine synthesis

209869993

2

8

5-aminoimidazole-4-carboxamide

purine synthesis

20127454

5

35

ribonucleotide formyltransferase adenylosuccinate lyase isoform a

purine synthesis

4557269

0

5

inosine monophosphate dehydrogenase 2

purine synthesis

66933016

1

14

phosphoribosylformylglycinamidine synthase

purine metabolism

31657129

3

14

guanine monophosphate synthetase

purine metabolism

4504035

1

12

hypoxanthine phosphoribosyltransferase 1 carbamoylphosphate synthetase 2

purine salvage pyrimidine synthesis

4504483 18105007

3 0

13 3

dihydropyrimidinase-like 3

pyrimidine metabolism

4503379

0

20

adenylosuccinate synthase

AMP synthesis

34577063

1

8

NME1-NME2 protein

CTP, GTP, UTP synthesis

66392203

11

32

adenylate kinase 2 isoform b

ATP/ADP ratio

deoxyuridine triphosphatase isoform 1 precursor

dUTP metabolism

7524346

3

12

70906441

1

11

acetyl-Coenzyme A acetyltransferase 2

mevalonate pathway

148539872

0

3

24-dehydrocholesterol reductase precursor cytochrome b5 outer mitochondrial membrane precursor

cholesterol synthesis cholesterol synthesis

13375618 83921614

0 1

5 7

aldehyde dehydrogenase 1A2 isoform 1

retinoic acid synthesis

25777724

3

29

uroporphyrinogen decarboxylase

porphyrin synthesis

71051616

0

4

branched chain aminotransferase 1, cytosolic

amino acid synthesis

38176287

0

12

ornithine aminotransferase precursor

proline synthesis

4557809

0

5

glutamine-fructose-6-phosphate transaminase 2

glutamate metabolism

4826742

0

3

methylenetetrahydrofolate dehydrogenase 1

one carbon metabolism

222136639

1

13

serine hydroxymethyltransferase 2 (mitochondrial) adenosylhomocysteinase isoform 1

one carbon metabolism one carbon metabolism

19923315 9951915

3 12

10 27

UDP-glucose dehydrogenase

nucleotide-sugar synthesis

36

NADP-dependent leukotriene B4 12-hydroxydehydrogenase

leukotriene metabolism

class I alcohol dehydrogenase, gamma subunit

ethanol catabolism

aldo-keto reductase family 1, member B10

aliphatic and aromatic

4507813

8

28570172

0

4

4501933

4

15

223468663

0

39

aldehydes reduction a

Functional annotation is based on Protein Knowledgebase at http://www.uniprot.org.

The LTQ-Orbitrap mass spectrometer was operated in a datadependent mode in which each full MS scan (60 000 resolving power) was followed by eight MS/MS scans where the eight most abundant molecular ions were dynamically selected and fragmented by collision-induced dissociation (CID) using a normalized collision energy of 35%. The Dynamic Exclusion Time was 30 s, and the Dynamic Exculsion Size was 200. The “FT master scan

preview mode”, “Charge state screening”, “Monoisotopic precursor selection”, and “Charge state rejection” were enabled so that only the 1þ, 2þ, and 3þ ions were selected and fragmented by CID. 2.4. Mass Spectrometry Data Analysis

Tandem mass spectra collected by Xcalibur (version 2.0.2) were searched against the NCBI human protein database using 1946



ARTICLE

Table 2. A Partial List of Down-Regulated Metabolic Proteins in PDAC functional annotationa

proteins

accession number

spectra count

spectra count

in normal duct

in PDAC

pyruvate dehydrogenase E1 alpha 1 precursor

Krebs cycle

4505685

8

0

pyruvate dehydrogenase (lipoamide) beta

Krebs cycle

156564403

11

1

aconitase 2, mitochondrial precursor

Krebs cycle

4501867

32

2

isocitrate dehydrogenase 2 (NADPþ), mitochondrial precursor

Krebs cycle

28178832

41

0

succinate-CoA ligase, GDP-forming alpha subunit

Krebs cycle

109452591

5

1

succinate-CoA ligase, GDP-forming beta subunit precursor

Krebs cycle

157779135

14

0

succinate dehydrogenase complex, subunit A, flavoprotein precursor succinate dehydrogenase complex, subunit B, iron sulfur (Ip) precursor

Krebs cycle Krebs cycle

156416003 115387094

17 7

5 0

NADH dehydrogenase ubiquinone flavoprotein 1 precursor

oxidative phosphorylation

20149568

5

0

NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75 kDa precursor


33519475

8

0

NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9, 39 kDa


6681764

6

0

ubiquinol-cytochrome c reductase core protein I


46593007

17

0

ubiquinol-cytochrome c reductase core protein II


50592988

13

0

cytochrome c-1


21359867

8

0

cytochrome c oxidase subunit II cytochrome c oxidase subunit VIb polypeptide 1

oxidative phosphorylation oxidative phosphorylation

17981856 4502985

5 7

0 2

ATP synthase, Hþ transporting, mitochondrial F0 complex,


21361565

12

3

fatty acid β -oxidation

76496475

32

2

subunit B1 precursor acyl-Coenzyme A dehydrogenase, very long chain isoform 2 precursor medium-chain acyl-CoA dehydrogenase isoform a precursor


4557231

7

0

short-chain acyl-CoA dehydrogenase precursor


4557233

8

2

mitochondrial trifunctional protein, alpha subunit precursor mitochondrial trifunctional protein, beta subunit precursor

fatty acid β -oxidation fatty acid β -oxidation

20127408 4504327

37 20

3 1 2

2,4-dienoyl CoA reductase 1 precursor


4503301

10

L-3-hydroxyacyl-Coenzyme A dehydrogenase precursor


94557308

9

0

acetyl-coenzyme A acyltransferase 2


167614485

25

0

UDP-glucose pyrophosphorylase 2 isoform a

glycogenesis

48255966

11

0

mitochondrial phosphoenolpyruvate carboxykinase 2

gluconeogenesis

66346721

7

0

isoform 1 precursor

a

fructose-1,6-bisphosphatase 1 aldo-keto reductase family 1, member A1

gluconeogenesis glucose metabolism

189083692 5174391

5 18

0 2

methylcrotonoyl-Coenzyme A carboxylase 2 (beta)

leucine degradation

11545863

13

1

3-hydroxy-3-methylglutaryl CoA lyase

leucine degradation

62198232

5

0

acetyl-Coenzyme A acetyltransferase 1 precursor

mevalonate pathway

4557237

25

5

nicotinamide phosphoribosyltransferase precursor

NADþ biosynthesis

5031977

25

0

L-arginine:glycine

creatine biosynthesis

4503933

25

0

amidinotransferase precursor

phosphoglycerate dehydrogenase

serine biosynthesis

23308577

22

0

dodecenoyl-Coenzyme A delta isomerase precursor hydroxysteroid (17-beta) dehydrogenase 10 isoform 1

fatty acid metabolism steroidogenesis

62530384 4758504

8 17

0 5

catechol-O-methyltransferase isoform MB-COMT

catecholamines degradation

brain creatine kinase

regeneration of ATP

4502969

7

0

21536286

13

0

adenylate kinase 1

ATP/ADP ratio

4502011

9

3

adenylate kinase 3

ATP/ADP ratio

19923437

9

2

manganese superoxide dismutase isoform A precursor

radicals removal

67782305

168

2

peroxiredoxin 4

antioxidant

5453549

23

5

peroxiredoxin 2 isoform a glutathione reductase

antioxidant antioxidant

32189392 50301238

16 16

3 4

carbonic anhydrase II

CO2 conversion

4557395

28

0

Functional annotation is based on Protein Knowledgebase at http://www.uniprot.org.

SEQUEST (Bioworks software from ThermoFisher, version 3.3.1) with full tryptic cleavage constraints, static cysteine

alkylation by iodoacetamide, and variable methionine oxidation. Mass tolerance for precursor ions was 5 ppm and mass tolerance 1947


Journal of Proteome Research for fragment ions was 0.25 Da. The SEQUEST search results of proteomics data were filtered by the criteria “Xcorr versus charge 1.9, 2.2, 3.0 for 1þ, 2þ, 3þ ions; ΔCn > 0.1; probability of randomized identification of peptide

Proteomic Analysis of Pancreatic Ductal Adenocarcinoma Cells

Recommend Documents