Open Access

Proteomic analysis of plasma membranes isolated from undifferentiated and differentiated HepaRG cells

  • Izabela Sokolowska1,
  • Cristina Dorobantu2,
  • Alisa G Woods1,
  • Alina Macovei2,
  • Norica Branza-Nichita2Email author and
  • Costel C Darie1Email author
Contributed equally
Proteome Science201210:47

DOI: 10.1186/1477-5956-10-47

Received: 9 February 2012

Accepted: 27 April 2012

Published: 2 August 2012

Abstract

Liver infection with hepatitis B virus (HBV), a DNA virus of the Hepadnaviridae family, leads to severe disease, such as fibrosis, cirrhosis and hepatocellular carcinoma. The early steps of the viral life cycle are largely obscure and the host cell plasma membrane receptors are not known. HepaRG is the only proliferating cell line supporting HBV infection in vitro, following specific differentiation, allowing for investigation of new host host-cell factors involved in viral entry, within a more robust and reproducible environment. Viral infection generally begins with receptor recognition at the host cell surface, following highly specific cell-virus interactions. Most of these interactions are expected to take place at the plasma membrane of the HepaRG cells. In the present study, we used this cell line to explore changes between the plasma membrane of undifferentiated (−) and differentiated (+) cells and to identify differentially-regulated proteins or signaling networks that might potentially be involved in HBV entry. Our initial study identified a series of proteins that are differentially expressed in the plasma membrane of (−) and (+) cells and are good candidates for potential cell-virus interactions. To our knowledge, this is the first study using functional proteomics to study plasma membrane proteins from HepaRG cells, providing a platform for future experiments that will allow us to understand the cell-virus interaction and mechanism of HBV viral infection.

Keywords

Hepatocytes HBV Proteomics Mass spectrometry Differentiation

Background

The hepatitis B virus (HBV) is a noncytopathic, hepatotropic DNA virus of the Hepadnaviridae family [1]. Infection with this virus leads to severe liver damage, such as fibrosis, cirrhosis and hepatocellular carcinoma [2]. Despite the existence of an efficient vaccine, more than 350 million people are currently HBV carriers at risk for developing life-threatening diseases.

While our understanding of HBV replication and assembly has advanced considerably in the last years, the early steps of the viral life cycle are still a matter of debate. This is mainly a consequence of the poor in vitro infectivity systems available, which until recently were based on primary human and chimpanzee hepatocytes [3]. Their accessibility is limited and the level of HBV replication is low, which makes the experimental data often difficult to interpret. The development of the HepaRG cell line, the only proliferating cells able to support the full HBV life cycle (following a specific differentiation treatment), unfolded new opportunities to investigate HBV infection in a more reproducible and reliable manner [4]. The ability of HepaRG to allow for HBV infection is reached only when cells are maintained quiescent at confluence and are treated with DMSO and hydrocortisone. While confluence alone is sufficient to activate many hepatic functions, DMSO treatment is compulsory for HBV productive infection. During differentiation, HepaRG cells express various liver functions in amounts comparable to those existing in primary hepatocytes [57]. Quantification of RNA levels within the whole population of differentiated cells showed high expression of adult hepatocytes-specific markers, such as albumin and aldolase B mRNAs, while the detoxification enzymes cytochrome P450, CYP 2E1 and CYP 3A4 were up-regulated in cells undergoing trabecular organization.

Generally, viral infection begins with receptor recognition and attachment to the host cell surface, followed by internalization of the virion by direct fusion at the plasma membrane, or endocytosis and later release from the endocytic vesicle. HBV appears to enter the target cells by receptor-mediated endocytosis, a process dependent on functional caveolin-1 expression [8]. Despite several potential cellular binding partners being reported to play a role in viral entry [4], none of these molecules was further confirmed to be the specific HBV receptor(s).

The rapid development of proteomics techniques has enabled the assessment of cellular proteins biosynthesis at a global scale, as well as the investigation of expression profile alterations under certain physiological or non-physiological conditions, with potential implications in cell function [911]. A previous proteomics study using HBV-uninfected and HBV-infected HepaRG cells identified 19 differentially-regulated proteins [12]. However, additional proteomic studies, more focused on plasma membrane proteins, (the first recognition partners during cell-virus interaction), are needed.

In the present study, we used the HepaRG cells to explore changes between the plasma membranes of undifferentiated (−) and differentiated (+) cells, and further identify differentially-regulated proteins that may potentially be involved in HBV entry or functional signaling networks that are activated upon cell-virus interaction. Our study identified a series of plasma-membrane-specific proteins, differentially expressed in (−) and (+) cells, with a potential role in viral infection. To our knowledge, this is the first study that focused on plasma membrane proteins from HePaRG cells using functional proteomics. The results obtained provide a platform for future investigations that will allow us to understand HBV cell-virus interactions and the molecular mechanisms of viral infection.

Results & discussion

Purification and verification of plasma membranes

Upon purification, we separated the plasma membranes from the (−) cells and (+) cells by SDS-PAGE, stained them by Coomassie dye and visually compared the protein pattern between the plasma membrane preparations from (−) and (+) cells. As observed, there is a clear difference between the protein patterns in these two preparations (Figure 1A). A difference in the intensity of the Coomassie-stained bands was also observed between (−) and (+) samples, despite an equal number of cells being used for plasma membrane preparation. Most probably this is a result of a better extraction of the transmembrane proteins from differentiated cells, as a consequence of an increased plasma membrane fluidity during prolonged treatment with 1.8% DMSO. This behavior is not unusual and was also observed during extraction of lipid raft proteins from differentiated HepaRG cells (data not shown) and is not directly related to the differentiation process.
https://static-content.springer.com/image/art%3A10.1186%2F1477-5956-10-47/MediaObjects/12953_2012_Article_384_Fig1_HTML.jpg
Figure 1

SDS-PAGE of the proteins from the plasma membranes isolated from the undifferentiated (−) and differentiated HepaRG cells. A: Coomassie stain of the SDS-PAGE gel showing the protein pattern for the plasma membrane of (−) and (+) cells. B: Expression of Cav-1, TRF-2 and calnexin in cell lysates (1) and plasma membrane fraction (2) of (−) cells was detected by Western blotting using the corresponding Abs. The molecular weight markers are indicated.

To confirm the plasma membrane isolation, total cell lysates, as well as a fraction of the (−) sample, were separated by SDS-PAGE and further analyzed by WB using antibodies against proteins with known plasma membrane or intracellular organelles localization. As observed in Figure 1B, expression of caveolin-1 (Cav-1) and transferrin receptor-2 (TRF-2) was detected in both, cell lysates and plasma membrane fraction, while the endoplasmic reticulum (ER) transmembrane protein, calnexin, was absent in the latter.

The latest investigations on HepaRG show that the number of differentiated cells, following DMSO treatment, is reasonably high (>50% of the total cell population) [13]. The significant up-regulation of hepatocyte-specific markers, considering the whole cell population, was clearly possible, ever since the cell line was described [4]. Thus, it is conceivable that changes of the level of expression (or post-translational modifications) of other proteins can be monitored in these cells.

LC-MS/MS identification of plasma membrane proteins

To further identify the proteins from the plasma membranes of the (−) and (+) cells, we cut bands out of the gel, digested them with trypsin and then analyzed them by LC/MS/MS. We performed two independent experiments, from two different preparations. Overall, we identified more proteins in the plasma membranes of the (+) cells, compared with the (−) cells. The results were consistent in both experiments. The outcome of two independent experiments is shown in Figure 2. Here are presented only the proteins identified with a Mascot score higher than 40. Also, the unnamed protein products, keratins and structural proteins (actin, tubulin) were removed from the final number of proteins presented in Figure 2. In experiment 1, we identified 118 proteins in the plasma membranes from (+) cells and 36 proteins in the plasma membranes from (−) cells. In this experiment, there was very little overlap between the two conditions (7 proteins). Similar results were observed in experiment 2: we identified 108 proteins in the plasma membranes from (+) cells and 25 proteins in the plasma membranes from (−) cells. The overlap between the two conditions was 10 proteins. The differentially identified proteins (proteins found only (−) but not in (+) and vice-versa) are presented in Tables 1 and 2. The complete lists with the proteins identified in our experiments are summarized in Additional file 1: Table S1 and Additional file 2: Table S2.
https://static-content.springer.com/image/art%3A10.1186%2F1477-5956-10-47/MediaObjects/12953_2012_Article_384_Fig2_HTML.jpg
Figure 2

Proteomic analysis of the proteins from (−) and (+) cells. The Coomassie-stained SDS-PAGE gel (as the one shown in Figure 1A) was divided in gel pieces and then subjected to LC-MS/MS analysis, as described in the methods section. Venn diagrams in A &B show the number of proteins identified per condition (from both (−) and (+) cells), as well as the number of proteins that are common or different in the (−) and (+) cells. Here we show the results from experiment 1 in A and experiment 2 in B.

Table 1

List with the proteins that were differentially identified by LC-MS/MS analysis of the plasma membranes of undifferentiated (−) and differentiated (+) cells (experiment #1)

 

Proteins identified only in (-) cells

Protein Accession #

Protein_description

gi|28614

aldolase A [Homo sapiens]

gi|134665

RecName: Full=superoxidase dismutase [Mn], mitochondrial, Flags: prescursor

gi|179930

carboxylesterase [Homo sapiens]

gi|2906146

malate dehydrogenase precursor [Homo sapiens]

gi|31645

glyceraldehyde-3-phosphate dehydrogenase [Homo sapiens]

gi|205830271

RecName: Full=Putative annexin A2-like protein; AltName: Full=Annexin A2 pseudogene 2

gi|30841303

manganese-containing superoxide dismutase [Homo sapiens]

gi|40068518

6-phosphogluconate dehydrogenase, decarboxylating [Homo sapiens]

gi|15012080

ACAT1 protein - Acetyl-CoA acetyltransferase [Homo sapiens]

gi|1723158

dihydrodiol dehydrogenase/bile acid-binding protein [Homo sapiens]

gi|134133226

POTE ankyrin domain family member E [Homo sapiens]

gi|4502013

adenylate kinase 2, mitochondrial isoform a [Homo sapiens]

gi|312137

fructose bisphosphate aldolase [Homo sapiens]

gi|5802974

thioredoxin-dependent peroxide reductase, mitochondrial isoform a precursor [Homo sapiens]

gi|257476

glutathione S-transferase A2 subunit [Homo sapiens]

gi|809185

Chain A, The Effect Of Metal Binding On The Structure Of Annexin V And Implications For Membrane Binding

gi|17389815

Triosephosphate isomerase 1 [Homo sapiens]

gi|511635

delta3, delta2-enoyl-CoA isomerase [Homo sapiens]

gi|56554357

Chain A, Binary Structure Of Human Decr Solved By Semet Sad.

gi|148733138

ubiquitously transcribed tetratricopeptide repeat protein Y-linked transcript variant 35 [Homo sapiens]

 

Proteins identified only in (+) cells

gi|30749518

Chain A, Crystal Structure Of Human Liver Carboxylesterase In Complex With Tacrine

gi|13937981

Peptidylprolyl isomerase A (cyclophilin A) [Homo sapiens]

gi|37267

transketolase [Homo sapiens]

gi|693933

2-phosphopyruvate-hydratase alpha-enolase [Homo sapiens]

gi|2981743

Chain A, Secypa Complexed With Hagpia (Pseudo-Symmetric Monomer)

gi|35505

pyruvate kinase [Homo sapiens]

gi|31645

glyceraldehyde-3-phosphate dehydrogenase [Homo sapiens]

gi|1633054

Chain A, Cyclophilin A Complexed With Dipeptide Gly-Pro

gi|860986

protein disulfide isomerase [Homo sapiens]

gi|291463384

Chain K, Acetyl-Cypa:cyclosporine Complex

gi|4557305

fructose-bisphosphate aldolase A isoform 1 [Homo sapiens]

gi|6009628

brain carboxylesterase hBr3 [Homo sapiens]

gi|999892

Chain A, Crystal Structure Of Recombinant Human Triosephosphate Isomerase At 2.8 Angstroms Resolution.

gi|306891

90kDa heat shock protein [Homo sapiens]

gi|4505591

peroxiredoxin-1 [Homo sapiens]

gi|61656603

Heat shock protein HSP 90 -alpha 2

gi|55959290

annexin A1 [Homo sapiens]

gi|1710248

protein disulfide isomerase-related protein 5 [Homo sapiens]

gi|5729877

heat shock cognate 71 kDa protein isoform 1 [Homo sapiens]

gi|62088648

tumor rejection antigen (gp96) 1 variant [Homo sapiens]

gi|4505763

phosphoglycerate kinase 1 [Homo sapiens]

gi|386758

GRP78 precursor [Homo sapiens]

gi|2906146

malate dehydrogenase precursor [Homo sapiens]

gi|34709

manganese superoxide dismutase (MnSOD) [Homo sapiens]

gi|32189394

ATP synthase subunit beta, mitochondrial precursor [Homo sapiens]

gi|4505753

phosphoglycerate mutase 1 [Homo sapiens]

gi|21361176

retinal dehydrogenase 1 [Homo sapiens]

gi|438069

thiol-specific antioxidant protein [Homo sapiens]

gi|178375

aldehyde dehydrogenase [Homo sapiens]

gi|930063

neurone-specific enolase [Homo sapiens]

gi|5453549

peroxiredoxin-4 precursor [Homo sapiens]

gi|11128019

cytochrome c [Homo sapiens]

gi|458470

carboxylesterase [Homo sapiens]

gi|1065111

Chain A, Nmr Structure Of Mixed Disulfide Intermediate Between Mutant Human Thioredoxin And A 13 amino acid Peptide

 

Comprising Its Target Site In Human Nfkb

gi|312137

fructose bisphosphate aldolase [Homo sapiens]

gi|913159

neuropolypeptide h3 [human, brain, Peptide, 186 aa]

gi|189238

neuroleukin [Homo sapiens]

gi|38648667

Fatty acid synthase [Homo sapiens]

gi|4503143

cathepsin D preproprotein [Homo sapiens]

gi|1065322

Chain A, A Surface Mutant (G82r) Of A Human Alpha-Glutathione S- Transferase

gi|6729803

Chain A, Heat-Shock 70kd Protein 42kd Atpase N-Terminal Domain

gi|189617

protein PP4-X [Homo sapiens]

gi|4758304

protein disulfide-isomerase A4 precursor [Homo sapiens]

gi|1040689

Human Diff6,H5,CDC10 homologue [Homo sapiens]

gi|544759

biliverdin-IX beta reductase isozyme I {EC 1.3.1.24} [human, liver, Peptide, 204 aa]

gi|62897129

heat shock 70kDa protein 8 isoform 1 variant [Homo sapiens]

gi|556516

dihydrodiol dehydrogenase isoform DD1 [Homo sapiens]

gi|1421609

Chain A, X-Ray Structure Of Nm23 Human Nucleoside Diphosphate Kinase B Complexed With Gdp At 2 Angstroms Resolution

gi|20070125

protein disulfide-isomerase precursor [Homo sapiens]

gi|729433

RecName: Full=Protein disulfide-isomerase A3; AltName: Full=58 kDa glucose-regulated protein; AltName:

gi|4757900

calreticulin precursor [Homo sapiens]

gi|5031857

L-lactate dehydrogenase A chain isoform 1 [Homo sapiens]

gi|1082886

tumor necrosis factor type 1 receptor associated protein TRAP-1 - human

gi|18204869

TUBA1B protein [Homo sapiens]

gi|5802966

destrin isoform a [Homo sapiens]

gi|119602577

plectin 1, intermediate filament binding protein 500kDa, isoform CRA_b [Homo sapiens]

gi|41322916

plectin isoform 1 [Homo sapiens]

gi|5174539

malate dehydrogenase, cytoplasmic isoform 2 [Homo sapiens]

gi|4503483

elongation factor 2 [Homo sapiens]

gi|3212355

Chain A, P11 (S100a10), Ligand Of Annexin Ii

gi|188492

heat shock-induced protein [Homo sapiens]

gi|21465695

Chain A, Human 3alpha-Hsd Type 3 In Ternary Complex With Nadp And Testosterone

gi|179930

carboxylesterase [Homo sapiens]

gi|4758012

clathrin heavy chain 1 [Homo sapiens]

gi|35830

ubiquitin activating enzyme E1 [Homo sapiens]

gi|4507953

14-3-3 protein zeta/delta [Homo sapiens]

gi|662841

heat shock protein 27 [Homo sapiens]

gi|4505257

moesin [Homo sapiens]

gi|4507357

transgelin-2 [Homo sapiens]

gi|4506467

radixin [Homo sapiens]

gi|6457378

cytovillin 2 [Homo sapiens]

gi|4505751

profilin-2 isoform b [Homo sapiens]

gi|6063147

ezrin [Homo sapiens]

gi|61104911

heat shock protein 90Bb [Homo sapiens]

gi|325533983

Chain A, Crystal Structure Of The Globular Domain Of Human Calreticulin

gi|119590453

EDAR-associated death domain, isoform CRA_a [Homo sapiens]

gi|311772317

Chain A, Crystal Structure Of Human Nad Kinase

gi|4504067

aspartate aminotransferase, cytoplasmic [Homo sapiens]

gi|5630077

similar to ALR; similar to AAC51735 (PID:g2358287) [Homo sapiens]

gi|896473

iron-responsive regulatory protein/iron regulatory protein 1 [Homo sapiens]

gi|5803225

14-3-3 protein epsilon [Homo sapiens]

gi|4826643

annexin A3 [Homo sapiens]

gi|332841153

PREDICTED: heat shock protein 105 kDa isoform 1 [Pan troglodytes]

 

Proteins identified in(-) and (+) cells

gi|4757756

annexin A2 isoform 2 [Homo sapiens]

gi|181250

cyclophilin [Homo sapiens]

gi|291463380

Chain B, Free Acetyl-Cypa Orthorhombic Form

gi|215273984

RecName: Full=Putative inactive carboxylesterase 4; AltName: Full=Inactive carboxylesterase 1 pseudogene 1

gi|809185

Chain A, The Effect Of Metal Binding On The Structure Of Annexin V And Implications For Membrane Binding

gi|4507913

wiskott-Aldrich syndrome protein family member 1

gi|438069

thiol-specific antioxidant protein [Homo sapiens]

Table 2

List with the proteins that were differentially identified by LC-MS/MS analysis of the plasma membranes of undifferentiated (−) and differentiated (+) cells (experiment #2)

 

Proteins identified only in (-) cells

Protein Accession #

Protein_description

gi|458470

carboxylesterase [Homo sapiens]

gi|62088648

tumor rejection antigen (gp96) 1 variant [Homo sapiens]

gi|28614

aldolase A [Homo sapiens]

gi|18645167

Annexin A2 [Homo sapiens]

gi|31179

enolase [Homo sapiens]

gi|34707

Manganese superoxide dismutase [Homo sapiens]

gi|4502013

adenylate kinase 2, mitochondrial isoform a [Homo sapiens]

gi|178390

aldehyde dehydrogenase [Homo sapiens]

gi|4504505

peroxisomal multifunctional enzyme type 2 isoform 2 [Homo sapiens]

gi|4503285

aldo-keto reductase family 1 member C2 isoform 1 [Homo sapiens]

gi|1723158

dihydrodiol dehydrogenase/bile acid-binding protein [Homo sapiens]

gi|6009628

brain carboxylesterase hBr3 [Homo sapiens]

gi|386758

GRP78 precursor [Homo sapiens]

gi|4507913

wiskott-Aldrich syndrome protein family member

 

Proteins identified only in (+) cells

gi|306891

90kDa heat shock protein [Homo sapiens]

gi|1633054

Chain A, Cyclophilin A Complexed With Dipeptide Gly-Pro

gi|4507677

endoplasmin precursor [Homo sapiens]

gi|75766275

Chain A, Crystal Structure Of Human Cypa Mutant K131a

gi|61656603

Heat shock protein HSP 90 - alpha 2 [Homo sapiens]

gi|179930

carboxylesterase [Homo sapiens]

gi|2981743

Chain A, Secypa Complexed With Hagpia (Pseudo-Symmetric Monomer)

gi|4757756

annexin A2 isoform 2 [Homo sapiens]

gi|4826898

profilin-1 [Homo sapiens]

gi|342350777

Chain A, Human Annexin V With Incorporated Methionine Analogue Azidohomoalanine

gi|32189394

ATP synthase subunit beta, mitochondrial precursor [Homo sapiens]

gi|157833780

Chain A, Human Annexin V With Proline Substitution By Thioproline

gi|53791219

filamin A [Homo sapiens]

gi|31645

glyceraldehyde-3-phosphate dehydrogenase [Homo sapiens]

gi|14327942

HSP90B1 protein [Homo sapiens]

gi|28614

aldolase A [Homo sapiens]

gi|662841

heat shock protein 27 [Homo sapiens]

gi|999892

Chain A, Crystal Structure Of Recombinant Human Triosephosphate Isomerase At 2.8 Angstroms

 

Resolution.

gi|35505

pyruvate kinase [Homo sapiens]

gi|693933

2-phosphopyruvate-hydratase alpha-enolase [Homo sapiens]

gi|4503483

elongation factor 2 [Homo sapiens]

gi|5729877

heat shock cognate 71 kDa protein isoform 1 [Homo sapiens]

gi|4505591

peroxiredoxin-1 [Homo sapiens]

gi|6525069

tumor necrosis factor type 1 receptor associated protein [Homo sapiens]

gi|4502101

annexin A1 [Homo sapiens]

gi|189617

protein PP4-X [Homo sapiens]

gi|4758012

clathrin heavy chain 1 [Homo sapiens]

gi|34419635

70kDa heat shock protein 6 [Homo sapiens]

gi|4507357

transgelin-2 [Homo sapiens]

gi|577295

KIAA0088 [Homo sapiens]

gi|35830

ubiquitin activating enzyme E1 [Homo sapiens]

gi|6470150

BiP protein [Homo sapiens]

gi|5453790

nicotinamide N-methyltransferase [Homo sapiens]

gi|5803227

14-3-3 protein theta [Homo sapiens]

gi|38648667

Fatty acid synthase [Homo sapiens]

gi|31746

glutathione-insulin transhydrogenase (216 AA) [Homo sapiens]

gi|1421609

Chain A, X-Ray Structure Of Nm23 Human Nucleoside Diphosphate Kinase B Complexed With

 

Gdp At 2 Angstroms Resolution

gi|438069

thiol-specific antioxidant protein [Homo sapiens]

gi|61104911

heat shock protein 90Bb [Homo sapiens]

gi|4758304

protein disulfide-isomerase A4 precursor [Homo sapiens]

gi|5802974

thioredoxin-dependent peroxide reductase, mitochondrial isoform a precursor [Homo sapiens]

gi|3318841

Chain A, Horf6 A Novel Human Peroxidase Enzyme

gi|230867

Chain R, Twinning In Crystals Of Human Skeletal Muscle D- Glyceraldehyde-3-Phosphate

gi|230867

Dehydrogenase

gi|5031857

L-lactate dehydrogenase A chain isoform 1 [Homo sapiens]

gi|4507877

vinculin isoform VCL [Homo sapiens]

gi|1710248

protein disulfide isomerase-related protein 5 [Homo sapiens]

gi|5031635

cofilin-1 [Homo sapiens]

gi|83753119

Chain A, Crystal Structure Of Human Full-Length Vinculin (Residues 1- 1066)

gi|157879202

Chain B, Crystal Structures Of Native And Inhibited Forms Of Human Cathepsin D: Implications

 

For Lysosomal Targeting And Drug Design

gi|74722493

RecName: Full=Putative heat shock protein HSP 90-alpha A4; AltName: Full=Heat shock 90 kDa

gi|74722493

protein 1 alpha-like 2; AltName: Full=Heat shock protein 90-alpha D

gi|181250

cyclophilin [Homo sapiens]

gi|182855

80K-H protein [Homo sapiens]

gi|37267

transketolase [Homo sapiens]

gi|1065111

Chain A, High Resolution Solution Nmr Structure Of Mixed Disulfide Intermediate Between

 

Mutant Human Thioredoxin And A 13 Residue Peptide Comprising Its Target Site In Human Nfkb

gi|1477646

plectin [Homo sapiens]

gi|4507953

14-3-3 protein zeta/delta [Homo sapiens]

gi|2138314

lysyl hydroxylase isoform 2 [Homo sapiens]

gi|283807248

Chain A, Crystal Structure Analysis Of W21a Mutant Of Human Gsta1-1 In Complex With

 

S-Hexylglutathione

gi|6005942

transitional endoplasmic reticulum ATPase [Homo sapiens]

gi|1065322

Chain A, A Surface Mutant (G82r) Of A Human Alpha-Glutathione S- Transferase Shows Decreased

gi|1065322

Thermal Stability And A New Mode Of Molecular Association In The Crystal

gi|799177

100 kDa coactivator [Homo sapiens]

gi|4505753

phosphoglycerate mutase 1 [Homo sapiens]

gi|5107666

Chain A, Structure Of Importin Beta Bound To The Ibb Domain Of Importin Alpha

gi|158937236

puromycin-sensitive aminopeptidase [Homo sapiens]

gi|5825506

fatty acid synthase/estrogen receptor fusion protein [Homo sapiens]

gi|5257007

beta-cop homolog [Homo sapiens]

gi|896473

iron-responsive regulatory protein/iron regulatory protein 1 [Homo sapiens]

gi|34707

Manganese superoxide dismutase [Homo sapiens]

gi|4506715

40S ribosomal protein S28 [Homo sapiens]

gi|3212355

Chain A, P11 (S100a10), Ligand Of Annexin Ii

gi|1065361

Chain A, Human Adp-Ribosylation Factor 1 Complexed With Gdp, Full Length Non-Myristoylated

gi|159162145

Chain A, Rotamer Strain As A Determinant Of Protein Structural Specificity

gi|51247357

Chain A, Crystal Structure Of A Multiple Hydrophobic Core Mutant Of Ubiquitin

gi|215273984

RecName: Full=Putative inactive carboxylesterase 4; AltName: Full=Inactive carboxylesterase 1

gi|215273984

pseudogene 1; AltName: Full=Placental carboxylesterase 3; Short=PCE-3; Flags: Precursor

gi|4502013

adenylate kinase 2, mitochondrial isoform a [Homo sapiens]

gi|5803013

endoplasmic reticulum resident protein 29 isoform 1 precursor [Homo sapiens]

gi|229532

ubiquitin

gi|31615803

Chain A, Synthetic Ubiquitin With Fluoro-Leu At 50 And 67

gi|2627129

polyubiquitin [Homo sapiens]

gi|6755368

40S ribosomal protein S18 [Mus musculus]

gi|313103963

Chain D, Structure And Control Of The Actin Regulatory Wave Complex

gi|6114601

stromal antigen 3, (STAG3) [Homo sapiens]

gi|4588526

nuclear chloride channel [Homo sapiens]

 

Proteins identified in(-) and (+) cells

gi|32189394

ATP synthase subunit beta, mitochondrial precursor [Homo sapiens]

gi|31645

glyceraldehyde-3-phosphate dehydrogenase [Homo sapiens]

gi|1710248

protein disulfide isomerase-related protein 5 [Homo sapiens]

gi|215273984

RecName: Full=Putative inactive carboxylesterase 4; AltName: Full=Inactive carboxylesterase 1

gi|215273984

pseudogene 1; AltName: Full=Placental carboxylesterase 3; Short=PCE-3; Flags: Precursor

gi|4758304

protein disulfide-isomerase A4 precursor [Homo sapiens]

gi|4505591

peroxiredoxin-1 [Homo sapiens]

gi|303618

phospholipase C-alpha [Homo sapiens]

gi|5031857

L-lactate dehydrogenase A chain isoform 1 [Homo sapiens]

gi|5802974

thioredoxin-dependent peroxide reductase, mitochondrial isoform a precursor [Homo sapiens]

gi|306891

90kDa heat shock protein [Homo sapiens]

It has been documented that the plasma membrane is the entry point for viruses [1416]. Therefore, we looked in our experiments for proteins that are possible interaction partners with viruses. One example is the Annexin family of proteins. For example, Annexin A2 (gi 4757756), a calcium-regulated protein that binds to the plasma membrane, is usually a heterotetramer of two Annexin A2 proteins and two S100A10 (gi 3212355) proteins. We identified both Annexin A2 proteins and S100A10 in the plasma membrane of the (+) cells, but not from the (−) cells, confirming the established interaction between these 2 proteins. Since Annexin A2 already has a history of interacting with viruses [1719], this suggests that an interaction with HBV may well be possible.

Two other proteins, also from the Annexin family, identified in our experiments were Annexin A1 (gi 4502101) and Annexin A5 (gi 157833780). Annexin A1 is a known phospholipase A2 inhibitory protein, but is also predicted to interact with Annexin A2 and perhaps form a protein complex (Figure 3). However, Annexin A1 was identified only in the plasma membranes from (+) cells, but not from the (−) cells, suggesting that this protein may be specific for the plasma membrane of (+) cells. The other protein, Annexin A5 is not predicted to interact with any of the other Annexins. However, it is well documented that Annexin A5 is an interaction partner for HBV [20]. Examples of MSMS spectra that correspond to peptides that are part of Annexin A2, Annexin A1, S100A10 protein and Annexin A5 are shown in Figure 4.
https://static-content.springer.com/image/art%3A10.1186%2F1477-5956-10-47/MediaObjects/12953_2012_Article_384_Fig3_HTML.jpg
Figure 3

Map of possible interaction partners for Annexin A2, Annexin A2 and S100A10. A: Annexin A2 (ANXA2) is predicted to interact with S100A10 and form a heterotetramer. B: Annexin A1 is predicted to interact with Annexin A2, via S100A11, ACTB or via ILB and TNF.

https://static-content.springer.com/image/art%3A10.1186%2F1477-5956-10-47/MediaObjects/12953_2012_Article_384_Fig4_HTML.jpg
Figure 4

MS/MS spectra of precursor ions that correspond to peptides that are part of Annexin A2, Annexin A1, S100A10, or Annexin A5. (A) A triple-charged peak at m/z of 551.27 (expanded in the inbox) was fragmented by MS/MS and produced a MS/MS spectrum. Fragmentation of the peptide backbone in the MS/MS produced a series of b and y peaks, marked in the MS/MS. Data analysis of the b and y peaks from the MS/MS spectrum led to identification of a peptide with the sequence SALSGHLETVILGLLK, which was part of Annexin A2 protein. (B): A double-charged peak at m/z of 803.53 (expanded in the inbox) was fragmented by MS/MS and produced a MS/MS spectrum. Data analysis of the MS/MS spectrum led to identification of a peptide with the sequence ALTGHLEEVVLALLK, which was part of Annexin A1 protein. (C) A double-charged peak at m/z of 550.57 (expanded in the inbox) was fragmented by MS/MS and produced a MS/MS spectrum. Data analysis of this MS/MS spectrum led to identification of a peptide with the sequence FAGDKGYLTK, which was part of S100A10 protein. (D) A double-charged peak at m/z of 618.07 (expanded in the inbox) was fragmented by MS/MS and produced a MS/MS spectrum. Data analysis of this MS/MS spectrum led to identification of a peptide with the sequence SEIDLFNIRK, which was part of Annexin A5 protein.

To further confirm that Annexin 2 and S100A10 interact with each other and also to investigate the interaction partners of these proteins and of other Annexin proteins, we explored the protein-protein interactions using the Search Tool for the Retrieval of Interacting Genes (STRING), a software tool that identifies known (experimentally documented) and predicted (theoretical only and yet to be demonstrated by experiments) protein-protein interactions [2124]. As observed, we did identify Annexin A2 as an interaction partner for S100A10 protein. However, we also identified a connection between Annexin A2 and Annexin A1, via S100A11, ACTB or via ILB and TNF proteins. The predicted interaction partners for Annexins A1 & A2 are presented in Figure 3.

Differential distribution of the proteins from the plasma membranes of (−) and (+) cells

The differential distribution of the proteins found by SDS-PAGE and LC-MS/MS in the plasma membranes of both (−) and (+) cells was also evaluated by their relative abundance using label-free methods for relative quantitation. These proteins differed in their abundance by either an increase or decrease of their relative amounts, as determined by both Mascot score (Additional file 1: Table S1), emPAI score [25], or by comparison of the relative intensity of the precursor ions that correspond to peptides that were part of the same proteins and that were identified in both (−) and (+) cells. The relative quantitation of these proteins was mostly used to determine whether some proteins were indeed specific to the plasma membranes from (+) cells, but not (−) cells. Using Annexin proteins as example, we looked at both the Mascot scores and emPAI scores for these proteins, as well as for the number of peptides identified per protein per condition in the database search, as well as direct comparison of the intensities of the precursor ions that correspond to the same peptide and for which MS/MS was observed in the same protein in both (−) and (+) conditions. For example, in the plasma membranes of (+) cells we identified Annexin A2 isoform 2 based on the MS/MS of 22 peptide, a mascot score of 957 and an emPAI score of 3.01, as compared with identification of the same protein in the plasma membranes of (−) cells, where we identified the same protein based on the MS/MS of only 6 peptides, a mascot score of 213 and an emPAI score of 0.63. To confirm that the relative amounts of Annexin A2 in the plasma membranes from (+) cells, but not (−) cells are indeed different in these two conditions, as reflected by the number of peptide and by Mascot scores in each condition, we further compared the intensity of precursor ions for two different peptides that were part of the same Annexin A2 protein and that were identified in the plasma membrane of the (−) and (+) cells. The intensity scale for the spectra for these peaks was normalized to identical number of counts. This comparison is shown in Figure 5. As observed, for the peak with m/z of 612.01 (2+) that corresponds to a peptide with the amino acid sequence TPAQYDASELK, a higher amount of this peptide is observed in the plasma membranes from the (+) cells, compared with the (−) cells (Figure 5A). Similar results were also observed for the peak with m/z of 623.03 (2+) that corresponds to a peptide with the amino acid sequence TNQELQEINR (Figure 5B).
https://static-content.springer.com/image/art%3A10.1186%2F1477-5956-10-47/MediaObjects/12953_2012_Article_384_Fig5_HTML.jpg
Figure 5

Comparison of the intensities of MS spectra for peaks corresponding to peptides that are part of Annexin A2 protein from the plasma membranes of (−) and (+) cells, found by SDS-PAGE and MS. A: The peak with m/z of 612.01 (2+) that corresponds to peptide TPAQYDASELK has a higher intensity in the plasma membranes of (+) cells, compared with the (−) ones. B: The peak with m/z of 623.03 (2+) that corresponds to peptide TNQELQEINR has a higher intensity in the plasma membranes of (+) cells, compared with the (−) ones. The intensity scale for the spectra from both (−) and (+) cells for each individual peptide was identical.

Concluding remarks

In this study, we used the HepaRG cells to investigate the differences between the protein content of the plasma membranes from differentiated and undifferentiated cells. We aimed to identify functional signaling networks and plasma membrane molecules that are expressed in differentiated cells and may potentially be involved in HBV entry. Using a proteomics approach, we identified the differentially expressed proteins and also concluded that they may form protein complexes such as Annexin A2 and S100A10 protein heterotetramer, with potential implications in cell-virus interaction. This approach is not only a source of the proteins present in the plasma membranes of the (−) and (+) cells, but also a starting point for identification of post-translational modifications of these proteins, as well as for determination of stable and transient protein-protein interactions, specifically HepaRG cell - HBV proteins interactions.

Experimental design

Chemicals

All chemicals were purchased from Sigma-Aldrich, unless mentioned otherwise.

Cell culture and differentiation

HepaRG cells (a kind gift from Dr. David Durantel, INSERM U871, Lyon, France) were grown in T75 flasks, in William’s E medium (Gibco) supplemented with 10% FCS, 50 units/ml penicillin, 50 μg/ml streptomycin, 2 mM GlutaMAX, 5 μg/ml insulin, and 5 x 105 M hydrocortisone hemisuccinate, as described [4]. To induce differentiation, cells were maintained for 2 weeks in William’s complete medium, without splitting, followed by 2 weeks in the same medium containing 1.8% DMSO. The typical cell morphology associated with differentiation was constantly monitored under the microscope and the up-regulation of albumin and aldolase B mRNAs was confirmed at the end of the differentiation process.

Preparation of plasma membranes

Differentiated (+) and non-differentiated (−) HepaRG cells were amplified in T75cm2 flasks (6 for each condition). All further steps were performed at 4°C. Cells were washed twice with 5 ml buffer A (0.25 M sucrose, 1 mM EDTA and 20 mM Tricine, pH = 7.8) and gently scraped in 3 ml of the same buffer. The cells were pelleted by centrifugation at 1400xg for 5 min, resuspended in 1 ml buffer A and disrupted with 20 strokes in a Douncer homogenizer. The homogenate was centrifuged at 1000xg for 10 min. The post nuclear supernatant (PNS) was stored on ice, and the pellet was resuspended in 1 ml buffer A, re-homogenized with 20 strokes in a Douncer homogenizer, as above. The first and second PNS were combined and layered on top of a 23 ml of 30% Percoll in buffer A, followed by centrifugation at 28,000 rpm in a Beckman ultracentrifuge SW41Ti rotor, for 30 min. The plasma membrane fraction was visible as a ring at approximately 5.7 cm from the bottom of the tube. This was collected and the Percoll was removed by dilution in 9.5 ml cold phosphate buffer saline (PBS), followed by 2 h ultracentrifugation at 30,000 rpm, as above. The supernatant was concentrated on a 10 kDa cutt-of centricon (Millipore) to a final volume of 150 μl.

SDS-PAGE and Western blotting

The isolated membranes were solubilized in Laemmli sample buffer for 5 minutes at 95°C and separated by SDS-PAGE, followed by Coomassie blue staining. To validate the plasma membrane fraction purification, the proteins were also transferred to nitrocellulose membranes (GE Healthcare) using a semi-dry blotter (Millipore). The blots were incubated with goat anti-TFR-2, goat anti-Calnexin (both Santa Cruz Biotechnology, dilution 1/1000), or rabbit anti–Cav-1 (Cell Signaling Technology, dilution 1/1000) antibodies (Ab), respectively, followed by donkey anti-goat (Santa Cruz Biotechnology, dilution 1/2000) or goat anti-rabbit horseradish peroxidase (HRP) Ab (Pierce, dilution 1/1000). Proteins were detected using the ECL (GE Healthcare) or the SuperSignal West Femto maximum Sensitivity Substrate (Thermo Scientific) detection systems according to the manufacturers’ instructions

Protein digestion and peptide extraction

The Coomassie-stained SDS-PAGE gels were cut into 3 gel pieces for each condition (plasma membranes isolated from undifferentiated (−) and differentiated (+) cells), and then treated according to published protocols [2629]. Briefly, the gel pieces were washed in high purity HPLC grade water for 20 minutes under moderate shaking and then and cut into very small pieces. The gel pieces were then dehydrated by incubation for 20 minutes in 50 mM ammonium bicarbonate, 20 minutes in 50 mM ammonium bicarbonate/50% acetonitrile, and 20 minutes in 100% acetonitrile. These three steps were performed under moderate shaking at room temperature. After the last incubation step, the gel pieces were dried in a Speed-vac concentrator and then rehydrated with 50 mM ammonium bicarbonate. The washing procedure was repeated twice. The dried gel bands were then rehydrated with a solution containing 10 mM DTT and 50 mM ammonium bicarbonate and incubated for 45 minutes at 56°C. DTT solution reduced the disulfide bridges in the proteins from the gel. The DTT solution was then replaced by a solution containing 100 mM iodoacetamide and 50 mM ammonium bicarbonate and further incubated for 45 minutes in the dark, with occasional vortexing. In this step, the cysteine residues were irreversibly modified by iodoacetamide to form carbamydomethyl-cysteine. The initial washing procedure was then repeated one more time, and then the gel pieces were dried in the Speed-vac concentrator and then rehydrated using 10 ng/μL trypsin (this leaves proteins at the peptide backbone at the Arg and Lys residues) in 50 mM ammonium bicarbonate, and then incubated overnight at 37°C under low shaking. The resulting peptides were extracted from the gel pieces by incubation with 5% formic acid/50 mM ammonium bicarbonate/50% acetonitrile (twice) and with 100% acetonitrile (once) under moderate shaking. Solutions containing peptide mixture were then combined and then dried in a Speed-vac concentrator. The peptides were then solubilized in 20 μL of 0.1% formic acid/2% acetonitrile/HPLC water, placed in UPLC vials and further used for LC-MS/MS analysis.

LC-MS/MS

The peptides mixture was analyzed by reversed phase liquid chromatography (LC) and MS (LC-MS/MS) using a NanoAcuity UPLC (Micromass/Waters, Milford, MA) coupled to a Q-TOF Micro MS (Micromass/Waters, Milford, MA), according to published procedures [2931]. Briefly, the peptides were loaded onto a 100 μm x 10 mm nanoAquity BEH130 C18 1.7 μm UPLC column (Waters, Milford, MA) and eluted over a 60 minute gradient of 2–80% organic solvent (acetonitrile containing 0.1% formic acid) at a flow rate of 400 nL/min. The aqueous solvent was HPLC water containing 0.1% formic acid. The column was coupled to a Picotip Emitter Silicatip nano-electrospray needle (New Objective, Woburn, MA). MS data acquisition involved survey MS scans and automatic data dependent analysis (DDA) of the top three ions with the highest intensity ions with the charge of 2+, 3+ or 4+ ions. The MS/MS was triggered when the MS signal intensity exceeded 10 counts/second. In survey MS scans, the three most intense peaks were selected for collision-induced dissociation (CID) and fragmented until the total MS/MS ion counts reached 10,000 or for up to 6 seconds each. Preliminary experiments were performed using an Alliance 2695 HPLC (Waters Corp, Milford, MA) that was coupled to the same Q-TOF Micro MS (Micromass/Waters, Milford, MA) described above. The peptides mixture was loaded onto an XBridgeTM C18 3.5 μm, 2.1 x 100 mm column (Waters Corporation, Milford, MA) and eluted over a 60 minutes gradient of 2–100% acetonitrile containing 0.1% formic acid at a flow rate of 200 μL/min. The aqueous phase was HPLC water containing 0.1% formic acid. The MS parameters in these experiments were unchanged from the previously described settings, except the source (micro source instead of nanosource). The entire procedure used was previously described [29, 32]. Calibration was performed for both precursor and product ions using either 1 pmol or 100 fmol GluFib standard peptide (Glu1-Fibrinopeptide B) with the sequence EGVNDNEEGFFSAR, with the monoisotopic m/z of 1770.68. The precursor ion monitored was the double charged peak of GluFib, with m/z of 785.84.

Data processing and protein identification

The raw data were processed using ProteinLynx Global Server (PLGS, version 2.4) software with the following parameters: background subtraction of polynomial order 5 adaptive with a threshold of 30%, two smoothings with a window of three channels in Savitzky-Golay mode and centroid calculation of top 80% of peaks based on a minimum peak width of 4 channels at half height. The resulting pkl files were submitted for database search and protein identification to the public Mascot database search ( http://​www.​matrixscience.​com, Matrix Science, London, UK) using the following parameters: human databases from NCBI and SwissProt, parent mass error of 1.3 Da, product ion error of 0.8 Da, enzyme used: trypsin, one missed cleavage, and carbamidomethyl-Cysteine as fixed modification and Methionine oxidized as variable modification. To identify the false negative results, we used additional parameters such as different databases or organisms, a narrower error window for the parent mass error (1.2 and then 0.2 Da) and for the product ion error (0.6 Da), and up to two missed cleavage sites for trypsin. In addition, the pkl files were also searched against in-house PLGS database version 2.4 ( http://​www.​waters.​com) using searching parameters similar to the ones used for Mascot search. The Mascot and PLGS database search provided a list of proteins for each gel band. To eliminate false positive results, for the proteins identified by either one peptide or a mascot score lower than 50, we verified the MS/MS spectra that led to identification of a protein. The proteins identified in our experiments are presented in Additional file 1: Table S1 and Additional file 2: Table S2. These proteins were identified with a Mascot score higher than 40. The proteins identified with a Mascot score lower than 40 were not considered, but the data can be provided upon request. The MS/MS spectra that allowed identification of a protein based on only one peptide are provided in Additional file 3: Figure S1. The MS/MS spectra provided in Additional file 3: Figure S1 were identified in Mascot database search with a score of 50 or higher in (+) cells. For the (−) cells, all MS/MS spectra are shown.

Notes

Abbreviations

HepaRG cells: 

A hepatoma-derived cell line, HBV, hepatitis B virus

SDS-PAGE: 

Aodium dodecyl sulfate-polyacrylamide gel electrophoresis

WB: 

Western blotting

MS: 

Mass spectrometry

LC-MS/MS: 

Liquid chromatography mass spectrometry

TIC: 

Total ion current

m/z: 

Mass/charge

CID: 

Collision-induced dissociation.

Declarations

Acknowledgements

C.C.D. thanks Ms. Laura Mulderig and her colleagues (Waters Corporation) for their generous support in setting up the Proteomics Center within the Biochemistry & Proteomics Group at Clarkson University. C.C.D. also thanks Dr. Thomas A. Neubert, Skirball Institute of Biomolecular Medicine, New York University for donation of the TofSpec2E MALDI-MS. This work was supported in part by Clarkson University (start-up to C.C.D.) and by the Army Research Office through the Defense University Research Instrumentation Program (DURIP grant #W911NF-11-1-0304 to C.C.D.). Drs. Norica Nichita, Alina Macovei and PhD student Cristina Dorobantu were supported by the Romanian Academy project 3 of the Institute of Biochemistry and the POSDRU/89/1.5/S/60746 grant.

Authors’ Affiliations

(1)
Biochemistry & Proteomics Group, Department of Chemistry & Biomolecular Science, Clarkson University
(2)
Department of Glycoproteins, Institute of Biochemistry of the Romanian Academy

References

  1. Chisari FV, Ferrari C: Hepatitis B virus immunopathogenesis. Annu Rev Immunol 1995, 13: 29–60.View Article
  2. Ganem D, Prince AM: Hepatitis B virus infection–natural history and clinical consequences. N Engl J Med 2004,350(11):1118–1129.View Article
  3. Glebe D, Urban S: Viral and cellular determinants involved in hepadnaviral entry. World J Gastroenterol 2007,13(1):22–38.PubMed CentralView Article
  4. Gripon P, Rumin S, et al.: Infection of a human hepatoma cell line by hepatitis B virus. Proc Natl Acad Sci U S A 2002,99(24):15655–15660.PubMed CentralView Article
  5. Aninat C, Piton A, et al.: Expression of cytochromes P450, conjugating enzymes and nuclear receptors in human hepatoma HepaRG cells. Drug Metab Dispos 2006,34(1):75–83.View Article
  6. Guillouzo A, Corlu A, et al.: The human hepatoma HepaRG cells: a highly differentiated model for studies of liver metabolism and toxicity of xenobiotics. Chem Biol Interact 2007,168(1):66–73.View Article
  7. Turpeinen M, Tolonen A, et al.: Functional expression, inhibition and induction of CYP enzymes in HepaRG cells. Toxicol In Vitro 2009,23(4):748–753.View Article
  8. Macovei A, Radulescu C, et al.: Hepatitis B virus requires intact caveolin-1 function for productive infection in HepaRG cells. J Virol 2010,84(1):243–253.PubMed CentralView Article
  9. Pandey A, Mann M: Proteomics to study genes and genomes. Nature 2000,405(6788):837–846.View Article
  10. Mann M, Hendrickson RC, et al.: Analysis of proteins and proteomes by mass spectrometry. Annu Rev Biochem 2001, 70: 437–473.View Article
  11. Aebersold R, Mann M: Mass spectrometry-based proteomics. Nature 2003,422(6928):198–207.View Article
  12. Narayan R, Gangadharan B, et al.: Proteomic analysis of HepaRG cells: a novel cell line that supports hepatitis B virus infection. J Proteome Res 2009,8(1):118–122.View Article
  13. Schulze A, Mills K, et al.: Hepatocyte polarization is essential for the productive entry of the hepatitis B virus. Hepatology 2012,55(2):373–383.View Article
  14. Caffrey M: HIV envelope: challenges and opportunities for development of entry inhibitors. Trends Microbiol 2011,19(4):191–197.PubMed CentralView Article
  15. Connolly SA, Jackson JO, et al.: Fusing structure and function: a structural view of the herpesvirus entry machinery. Nat Rev Microbiol 2011,9(5):369–381.PubMed CentralView Article
  16. Cosset FL, Lavillette D: Cell entry of enveloped viruses. Adv Genet 2011, 73: 121–183.View Article
  17. Raynor CM, Wright JF, et al.: Annexin II enhances cytomegalovirus binding and fusion to phospholipid membranes. Biochemistry 1999,38(16):5089–5095.View Article
  18. Rai T, Mosoian A, et al.: Annexin 2 is not required for human immunodeficiency virus type 1 particle production but plays a cell type-dependent role in regulating infectivity. J Virol 2010,84(19):9783–9792.PubMed CentralView Article
  19. Yang SL, Chou YT, et al.: Annexin II binds to capsid protein VP1 of enterovirus 71 and enhances viral infectivity. J Virol 2011,85(22):11809–11820.PubMed CentralView Article
  20. Hertogs K, Leenders WP, et al.: Endonexin II, present on human liver plasma membranes, is a specific binding protein of small hepatitis B virus (HBV) envelope protein. Virology 1993,197(2):549–557.View Article
  21. Snel B, Lehmann G, et al.: STRING: a web-server to retrieve and display the repeatedly occurring neighbourhood of a gene. Nucleic Acids Res 2000,28(18):3442–3444.PubMed CentralView Article
  22. von Mering C, Jensen LJ, et al.: STRING 7--recent developments in the integration and prediction of protein interactions. Nucleic Acids Res 2007,35(Database issue):D358-D362.PubMed CentralView Article
  23. Jensen LJ, Kuhn M, et al.: STRING 8--a global view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res 2009,37(Database issue):D412-D416.PubMed CentralView Article
  24. Szklarczyk D, Franceschini A, et al.: The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res 2011,39(Database issue):D561-D568.PubMed CentralView Article
  25. Ishihama Y, Oda Y, et al.: Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol Cell Proteomics 2005,4(9):1265–1272.View Article
  26. Shevchenko A, Wilm M, et al.: Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 1996,68(5):850–858.View Article
  27. Darie CC, Biniossek ML, et al.: Structural characterization of fish egg vitelline envelope proteins by mass spectrometry. Biochemistry 2004,43(23):7459–7478.View Article
  28. Darie CC, Biniossek ML, et al.: Mass spectrometric evidence that proteolytic processing of rainbow trout egg vitelline envelope proteins takes place on the egg. J Biol Chem 2005,280(45):37585–37598.View Article
  29. Sokolowska I, Woods AG, Wagner J, Dorler J, Wormwood K, Thome J, et al.: Mass Spectrometry for Proteomics-based Investigation of Oxidative Stress and Heat Shock Proteins . In Oxidative Stress: Diagnostics, Prevention and Therapy. Washington: ACS Symposium Miniseries, ACS Publications; 2011:369–411.View Article
  30. Spellman DS, Deinhardt K, et al.: Stable isotopic labeling by amino acids in cultured primary neurons: application to brain-derived neurotrophic factor-dependent phosphotyrosine-associated signaling. Mol Cell Proteomics 2008,7(6):1067–1076.PubMed CentralView Article
  31. Darie CC, Deinhardt K, Zhang G, Cardasis HS, Chao MV, Neubert TA: Identifying transient protein-protein interactions in EphB2 signaling by blue native PAGE and mass spectrometry. Proteomics 2011,11(23):4514–4528.PubMed CentralView Article
  32. Sokolowska I, Woods AG, Gawinowicz MA, Roy U, Darie CC: Identification of potential tumor differentiation factor (TDF) receptor from steroid-responsive and steroid-resistant breast cancer cells. [Research Support, Non-U.S. Gov't]. J Biol Chem 2012,287(3):1719–1733.PubMed CentralView Article

Copyright

© Sokolowska et al.; licensee BioMed Central Ltd. 2012

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Advertisement