Quantitative Proteomics by Amino Acid Labeling in Foot-and-Mouth

Article pubs.acs.org/jpr

Quantitative Proteomics by Amino Acid Labeling in Foot-and-Mouth Disease Virus (FMDV)-Infected Cells Yu Ye,†,‡,§ Guangrong Yan,⊥ Yongwen Luo,‡ Tiezhu Tong,∥ Xiangtao Liu,# Chaoan Xin,†,‡,§ Ming Liao,*,†,‡,§ and Huiying Fan*,†,‡,§ †

Key Laboratory of Animal Vaccine Development, Ministry of Agriculture, Guangzhou 510642, China College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China § National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control, Guangzhou 510642, China ∥ Huizhou Entry-Exit Inspection and Quarantine Bureau, Huizhou 516001, China ⊥ Institute of Life and Health Engineering and National Engineering and Research Center for Genetic Medicine, Jinan University, Guangzhou 510632, China # State Key Laboratory of Veterinary Etiologic Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China ‡

S Supporting Information *

ABSTRACT: Foot-and-mouth disease virus (FMDV) is an important disease agent that can be difficult to effectively eradicate from herds. Because it is an obligate intracellular parasite, the virus has multiple effects on the host cell during infection. Here, a high-throughput quantitative proteomic approach was used to develop an unbiased holistic overview of the protein changes in IBRS-2 cells infected with FMDV. Stable isotope labeling with amino acids in cell culture (SILAC) combined with LC−MS/MS was performed to identify and quantify 1260 cellular and 2 viral proteins after 6 h of infection of IBRS-2 cells with FMDV. Of these identified and measured cellular protein pairs, 77 were significantly up-regulated, and 50 were significantly down-regulated based on significance B ≤ 0.05. The differentially altered proteins included a number of proteins involved in endolysosomal proteases system, cell cycle, cellular growth and proliferation, and immune cell trafficking. Selected data were validated by Western blot. Ingenuity Pathway Analysis revealed that proteins that changed in response to infection could be assigned to defined canonical pathways and functional groupings, such as integrin signaling. The obtained data might not only improve the understanding of the dynamics of FMDV and host interaction but may also help elucidate the pathogenic mechanism of FMDV infection. KEYWORDS: foot-and-mouth disease virus (FMDV), stable isotope labeling with amino acids in cell culture (SILAC), quantitative proteomics, pathway analysis

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INTRODUCTION

controlling and preventing dissemination of this severe epidemic in many regions of the world is an unsolved problem. FMDV is the prototypic member of Aphthovirus genus in the Picornaviridae family. Its genome consists of a single-stranded positive sense RNA of approximately 8.3 kb in length. The genomic RNA contains a single open reading frame (ORF), flanked by highly structured 5′ and 3′ untranslated regions (5′ UTR and 3′ UTR, respectively). The viral RNA is translated into one large polyprotein through the classical capindependent translation initiation mechanism via an internal ribosomal entry site (IRES) located in the 5′ UTR. The polyprotein is posttranslationally cleaved by viral proteinases

Foot-and-mouth disease is one of the world's major uncontrolled pathogens, and it is responsible for huge global losses in livestock production and trade. The causal agent, footand-mouth disease virus (FMDV), is a nonenveloped RNA virus that causes a highly contagious disease affecting up to 70 species of cloven-hoofed mammals. The disease is characterized by fever, nasal discharge, and lesions on the tongue and/or feet; the mortality rate is high in young animals.1 FMDV is virulent and has multiple known routes of transmission; the airborne pathway is suspected to play a key role in some outbreaks by causing disease “sparks”.2 There are seven immunologically distinct serotypes: O, A, C, SAT 1, SAT 2, SAT 3, and Asia 1; there are more than 60 strains within each of these serotypes. New mutant strains occasionally develop spontaneously. So far, © 2012 American Chemical Society

Received: July 7, 2012 Published: November 21, 2012 363

dx.doi.org/10.1021/pr300611e | J. Proteome Res. 2013, 12, 363−377

Journal of Proteome Research

Article

into individual structural and nonstructural proteins.3 Four structural proteins, 1A, 1B, 1C, and 1D (also known as VP4, VP2, VP3, and VP1, respectively), have been identified to form the natural empty capsid, and 60 copies of each protein will self-assemble to form a particle. Multiple antigenic sites, located at the surface of the viral particle, bind to integrin receptors and heparan sulfate proteoglycan receptors on the host cell surface to release viral RNA into the cytoplasm. Nonstructural proteins represent approximately two-thirds of proteins encoded by the ORF, including Lpro, 2A, 2B, 2C, 3A, 3B, 3Cpro, and 3Dpol.4 These proteins regulate viral replication, protein processing, and protein modification in the host. FMDV polyprotein is processed by a variety of virus-encoded proteases (Lpro, 2A, and 3Cpro). Lpro, a papain-like proteinase, mediates autocatalytic cleavage of itself from a polyprotein and cleaves the host protein eIF4GI and eIF4GII, resulting in the shut-off of host cap-dependent mRNA translation.5 3Cpro can efficiently process all 10 cleavage sites in the polyprotein, can cleave the host proteins eIF4A and eIF4G, and may inhibit host cell transcription by cleavage of histone H3.6 2A appears to function as an autoproteinase, permitting the synthesis of the downstream proteins at the level of the ribosome.7 2B and 2C proteins localize to the ER-derived outer surface vesicles, which are the sites of genome replication in the picornaviruses.8 3A is shown to colocalize with the intracellular membrane system where viral RNA replication occurs.9 3B (VPg) is covalently bound to the 5′ terminus of the genome and implicated in priming replication.10 3Dpol constitutes the FMDV RNAdependent RNA polymerase and is thus the catalytic core of the replicase, which interacts with 3A to form the viral replication complex.11,12 During the course of infection, FMDV attempts to subvert numerous mechanisms established by host cell functions that inhibit viral propagation. Previous studies have generally focused on defined cellular and viral pathways to elucidate the complex interplay between FMDV and host cells. Lpro inhibits the transcription of various cellular genes including interferon β (IFN-β) and tumor necrosis factor α (TNF-α),13 to antagonize the innate immune response through degrading NF-κB.14 Importantly, these actions result in the impaired ability of natural killer cells (NK cells) to recognize and eliminate FMDV-infected cells, allowing the virus to replicate and disseminate within the host.15 Lbpro, a shorter form of Lpro, mediates deubiquitination, apparently inhibiting retinoic acidinducible gene I (RIG-I), TANK-binding kinase 1 (TBK1), TNF receptor-associated factor 6 (TRAF6), and TRAF3, which is the key signal molecule in the activation of the type I IFN response.16 In addition to both modulating the antiviral response within infected cells and promoting long-term persistence of the virus, FMDV recruits various host cell factors to assist in the translation and replication of its genome. RNA helicase A plays an essential role in the replication of FMDV and potentially other picornaviruses through ribonucleoprotein complex formation at the 5′ end of the genome and by interactions with 2C, 3A, and PABP.17 Additionally, inhibition of the autophagic pathway decreases viral replication.18 To date, proteomics has not yet been applied to further analyze the whole cell changes caused by FMDV replication. Comprehensive knowledge of alterations in the amount of host cellular proteins during the infection of FMDV could provide valuable insight into the molecular mechanisms of viral replication and promote the development of a new generation

of drugs that targets host cell factors and are thus less prone to the selection of resistant viral mutants. To investigate whether the IBRS-2 cell proteome changed after FMDV infection, highthroughput quantitative proteomics, using stable isotope labeling with amino acids in cell culture (SILAC), was used in conjunction with liquid chromatography−mass spectrometry/mass spectrometry (LC−MS/MS). SILAC is a precise mass spectrometry-based quantitative strategy that provides a defined number of labels per identified peptide and therefore enables easier and more comprehensive peptide identification and data analysis.19 With this method, cellular and possibly viral proteins were identified and quantified. In total, 127 proteins of whose abundance was significantly altered (significance B ≤ 0.05) were identified in response to FMDV infection. Bioinformatics analyses of protein networks and pathways indicated that signaling cascades could be induced in FMDV-infected IBRS-2 cells. Proteins involved in the endolysosomal proteases system, cell cycle, cellular growth and proliferation, and immune cell trafficking were significantly changed.

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MATERIALS AND METHODS

Virus and Cells Porcine kidney (IBRS-2) cells were purchased from CCTCC (China Center for Type Culture Collection) and grown in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% dialyzed fetal calf serum and 1% penicillin−streptomycin. All cells were tested to ensure that there was no mycoplasma contamination. For the SILAC experiments, IBRS-2 cells were cultured in media either containing 13C- and 15N-labeled L-arginine and 2D-labeled L-lysine (R10K4) or containing unlabeled L-arginine and L-lysine amino acids (R0K0) for at least five cell doublings. FMDV strain O/QYYS/s/06 was isolated and preserved at Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, and propagated in R10K4labeled IBRS-2 cells. It is a member of the Cathay topotype virus, which is phylogenetically and evolutionally related to the strain O/ HKN/2002 (98%) and O/Taiwan/97 (92%) in full-length genomic sequence level.20 Virus titer was calculated based on plaque assay titration in IBRS-2 cells.

Infection Cells that were cultured in a conventional source of amino acids (R0K0) were used as mock-infected controls, while heavy-labeled (R10K4) cells were infected with FMDV. Cell batches that were passaged in the two media were seeded in 150 cm2 cell culture flasks until they reached 75% confluence. They were then inoculated with virus stock corresponding to a multiplicity of infection (MOI) of 1 (R10K4 -labeled cells), or they were mock inoculated (R0K0 -labeled cells). The amount of dialyzed serum in the SILAC media was decreased to 2%. The IBRS-2 cells were harvested at 6 h postinfection (h p.i.).

Preparation of Protein Samples and In-Gel Trypsin Digestion Cell pellets were resuspended in a cold lysis buffer and incubated for 10 min on ice. The lysate was sonicated for 10 cycles of 0.8 s on and 0.8 s off and then centrifuged to pellet the cellular debris. Protein quantification was performed using the Bradford protein assay with a 2-D Quant Kit (PlusOne). Prior to gel electrophoresis, equal amounts of protein extract from FMDV-infected cells and uninfected cells were mixed, separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and silver stained to visualize the gel bands. The gel lanes were cut horizontally into gel pieces that were then in-gel destained, reduced, alkylated, and digested with goldtrypsin at 37 °C overnight as described previously.21,22 The tryptic peptides were extracted, and the peptide mixtures were concentrated by SpeedVac centrifuge to dryness and redissolved with 2% acetonitrile (ACN) in 0.1% formic acid before LC−MS/MS analysis. 364



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Figure 1. Photomicrographs of IBRS-2 cells infected with FMDV strain O/QYYS/s/06 at MOI = 1 PFU/cell (bottom) or mock-infected (top) for the indicated hours postinfection (indicated at top). Images were taken at an original magnification of 100×.

LC−MS/MS Analysis

blocked with 5% nonfat dry milk in TBS containing 0.05% Tween20 (TBST) overnight at 4 °C and then incubated for 1 h at room temperature with rabbit polyclonal antibody against β-tubulin (Bioworlde Technology, Inc.) and mouse monoclonal antibodies Cathepsin D (Santa Cruz Biotechnology, Inc.) or Caplain-1 (Santa Cruz Biotechnology, Inc.). Membranes were washed in TBST and incubated with DyLight488-conjugated goat antirabbit immunoglobulin G (IgG) or rabbit antimouse IgG (1:10000, Rockland) for 1 h. Membranes were washed in TBST and visualized using the Odyssey Infrared Imaging system (Licor Biosciences, Lincoln, NE).

The peptide mixtures were separated by high-performance liquid chromatography (HPLC) (Agilent 1200) on a C18 reverse phase column and analyzed using an LTQ-Orbitrap mass spectrometer (Thermo Electron). The spray voltage was set to 1.85 kV, and the temperature of the heated capillary was set to 200 °C. Full-scan MS survey spectra (m/z 400−2000) in profile mode were acquired in the Orbitrap with a resolution of 60000 at m/z 400 after the accumulation of 1000000 ions. The five most intense peptide ions from the preview scan in the Orbitrap were fragmented by collision-induced dissociation (normalized collision energy, 35%; activation Q, 0.25; and activation time, 30 ms) in the LTQ after the accumulation of 5000 ions. Precursor ion charge state screening was enabled, and all unassigned charge states and singly charged species were rejected. The dynamic exclusion list was restricted to a maximum of 500 entries with a maximum retention period of 90 s and a relative mass window of 10 ppm. The data were acquired using Xcalibur software (Thermo Electron).

Photomicrography Infected and mock-infected cells in the six-well plates (NEST) were examined microscopically for a cytopathic effect (CPE) at 0, 2, 4, 6, and 8 h p.i. with a LEICA DMIL, and they were photographed with a Canon powershot S70 digital camera. Images were imported into Adobe, and slight adjustments were made in brightness and contrast, but these adjustments did not alter the relative context of the findings.

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Protein Identification, Quantification, and Bioinformatics Protein identification and quantitation were performed as previously described with minor modifications.23 In brief, raw MS spectra were processed by using MaxQuant 1.0.13.13 software, and the derived peak lists were searched using the Mascot 2.2.04 search engine (Matrix Science, London, United Kingdom) against a concatenated forwardreverse database from the National Center for Biotechnology information nonredundant (NCBinr) database (August 30, 2011) containing pig (sus scrofa) sequences. Parameters allowed included up to two missed cleavages and two labeled amino acids (arginine and lysine). The other search parameters were employed: full tryptic specificity was required, carbamidomethylation was set as a fixed modification, whereas oxidation (M) was considered a variable modification. Precursor ion mass tolerances were 10 ppm for all MS acquired in the Orbitrap mass analyzer, and the fragment ion mass tolerance was 0.5 Da for all MS2 spectra acquired in the LTQ. The minimum required peptide length was set to six amino acids. Mascot search results were further processed by the MaxQuant 1.0.13.13 program at a false discovery rate (FDR) of 1% at both the protein, the peptide, and the site level. The normalized H/L ratios, significance, and variability (%) were automatically calculated by the MaxQuant 1.0.13.13 program. The resulting proteingroups.txt output file from MaxQuant containing the peptide identifications was imported into Microsoft Excel, in which additional filtering was performed [protein posterior error probability (PEP) ≤0.01]. Gi numbers of all significantly regulated proteins were imported into the Ingenuity Pathway Analysis software (IPA, www.ingenuity.com) for bioinformatics analysis based on published reports and databases such as Gene Ontology and Uniport.

RESULTS

Kinetics of FMDV-Induced Cytopathology in Cultured IBRS-2 Cells

One of the key parameters for determining virus-induced alterations, and in separating such alterations from general stress responses related to cell death late in infection, is to determine when cytopathic effects (CPE) are manifested in the model system.24 Upon FMDV entry into a cell, the viral genome is rapidly translated by polysomes. For the prototype of FMDV type O, infection of host cells results in detectable viral early gene expression by 0.5 h p.i., viral RNA synthesis that was found to be almost complete at approximately 3 h p.i., production of structural proteins that will form progeny virions at approximately 4−6 h p.i., and release of viral particles and lysis of the infected cells from approximately 6 to 10 h onward.25 Accordingly, IBRS-2 cells were infected by FMDV strain O/QYYS/s/06 at a multiplicity of infection (MOI) of 1 and microscopically monitored for cell viability and cytopathic effect (CPE) over time. A minimal CPE became visible approximately 6 h p.i., and CPE was readily apparent at later time points (Figure 1). Two viral proteins were also identified in this analysis, ployprotein and VP1 (Table 1 in the Supporting Information). Therefore, in the subsequent study, we chose to examine the composition of cells at 6 h p.i., when structural proteins gene expression would be just underway in some cells but when active viral replication would be well underway in virtually all of the cells examined and exert maximal effect with minimal demonstrable CPE.22

Western Blot Analysis The protein concentrations of FMDV-infected and uninfected cell lysates, harvested at 6 h p.i., were measured. Equivalent amounts of cell lysates from two independent biological replicates were denatured in a 5× sample loading buffer by heating at 70 °C for 10 min and were then separated by 10% SDS-PAGE. Proteins were electrotransferred to 0.45 μm nitrocellulose membranes (Bio-Rad). Membranes were

SILAC Analyses of the Virus-Infected Cells Proteome

Until now, no previous study has used SILAC coupled with LC−MS/MS to identify and quantify proteome changes in cells infected with FMDV. In this study, we harvested cellular 365



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and disorders, molecular and cellular functions, physiological system development, and functions and toxicity functions that were identified at statistically significant levels (p < 0.05) are depicted in Figure 5, with additional data shown in Table 3 in the Supporting Information. The 77 up-regulated proteins, which correspond to 23 diseases and disorders (Figure 5A, left), included proteins that are related to cancer, genetic disorders, skeletal and muscular disorders, inflammatory disease, immunological disease, reproductive system disease, hematological disease, gastrointestinal disease, and neurological disease. These up-regulated proteins can also be assigned to 27 molecular and cellular functions groups (Figure 5B, left), including cellular growth and proliferation, cell death, cellular movement, molecular transport, protein synthesis, cell-to-cell signaling and interaction, and small molecule biochemistry; 25 physiological system development and functions groups (Figure 5C, left), including hematological system development and function, immune cell trafficking, tissue development, cardiovascular system development and function, embryonic development, organismal development, and connective tissue development and function; and 16 toxicity functions groups (Figure 5D, left), including hepatocellular carcinoma, renal necrosis/cell death, liver hepatitis, heart failure, nephrosis, and cardiac stenosis. The 50 down-regulated proteins are primarily related to 24 diseases and disorders (Figure 5A, right), including genetic disorders, cancer, neurological disease, dermatological diseases and conditions, inflammatory disease, gastrointestinal disease, hematological disease, infectious disease, and cardiovascular disease. These down-regulated proteins are also assigned to 28 molecular and cellular functions groups (Figure 5B, right), including small molecule biochemistry, protein synthesis, cell death, cell cycle, cellular growth and proliferation, cellular assembly and organization, lipid metabolism, cellular function and maintenance, and molecular transport; 19 physiological system development and functions groups (Figure 5C, right), including connective tissue development and function, tissue development, organ morphology, endocrine system development and function, hair and skin development and function, and reproductive system development and function; and 13 toxicity functions groups (Figure 5D, right), including hepatocellular carcinoma, cardiac hypertrophy, renal tubule injury, bradycardia, and glomerular injury. IPA was used to determine if proteins that changed in abundance could be mapped to specific functional networks and generate several biological pathways of interest that may be common to viral infection. For the whole-cell proteome, network analysis suggested proteins that alter significantly were mapped to eight specific functional networks (Figure 6), and each with 10 or more “focus” members shared common members (Table 3 in the Supporting Information). The other network (network 9) consisted of two proteins but contained only a single focus protein (Figure 6 and Table 3 in the Supporting Information). The four networks of interest correspond to (1) cellular movement, cell-to-cell signaling and interaction, and tissue development (Figure 7A); (2) lipid metabolism, small molecule biochemistry, and carbohydrate metabolism (Figure 7B); (3) cancer, respiratory disease, and gastrointestinal disease (Figure 7C); and (4) protein synthesis, post-translational modification, and cellular compromise (Figure 7D). Proteins that are present in these pathways and identified in our analysis as up-regulated are depicted in shades of red, and those that were identified as down-regulated are

proteome from infected and mock-infected FMDV cells and compared the differences in the isolated proteins by quantitative proteomics. In total, 7188 peptides and 1236 proteins were detected, and 6912 (96.16%) peptides and 1206 (97.57%) proteins could be quantified by LC−MS/MS analysis, respectively. Of these, 50 proteins were highly down-regulated, and 77 were significantly up-regulated, based on a significance of B ≤ 0.0519,23 (Figures 2 and 3 and Table 1).

Figure 2. Distributions of proteins identified in the experiment. Up represents up-regulated host proteins in virus-infected cells. Down represents down-regulated host proteins, based on significance B ≤ 0.05. The rest part represents unchanged proteins in FMDV-infected cells.

Subcellular and Functional Characterization and Bioinformatics Analysis of the IBRS-2 Cell Proteome

To gain functional insights into the cellular proteome, 127 identified proteins were assigned to different molecular functional classes and subcellular locations according to the underlying biology evidence from the UniProtKB/Swiss-Prot and the Gene Ontology database. Biological processes, molecular functions, and cellular components identified are shown in Table 2 in the Supporting Information. In Figure 4A, 50 down-regulated proteins in infected cells were localized to unknown cellular location (42.00%), nucleus and cytoplasm (22.00%), membrane (8.00%), extracellular space (8.00%), mitochondrion (8.00%), cytoplasm (6.00%), nucleus (4.00%), and cytoskeleton (2.00%). Sevety-seven up-regulated proteins (Figure 4B) were localized to unknown cellular location (29.87%), ribosome (24.68%), cytoplasm (9.09%), membrane (7.79%), extracellular space (7.79%), mitochondrion (5.19%), nucleus (3.90%), nucleus and cytoplasm (3.09%), membrane and cytoplasm (3.90%), cytoskeleton (2.60%), and cytoskeleton (1.30%). Because the pig genome database had poor annotation as compared to the human genome and many proteins were unassigned or uncharacterized, gene identifications of the identified proteins in Table 1 were converted to human protein gi numbers. Protein gi numbers and levels of regulation were imported into the IPA tool, and interacting pathways were constructed based on the underlying biological evidence from the literature database. The organization of previously characterized 127 proteins, which were grouped into several distinctive functional sets, reveals that the proteome differed from increases to decreases, which presented a variety of meaningful information. Diseases 366



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Figure 3. Proteome-wide accurate quantitation and significance. Signal intensities (log10) of all quantified proteins in the FMDV-infected experiment are shown as a function of their fold change (log2). The spread of the cloud is lower at high abundance, indicating that quantification is more precise. The criteria as being identified as a significantly regulated protein can be evaluated by the significance B level indicated in blue, red, yellow, and green, respectively.

shown in green. Proteins that are present in the pathways and identified in our analysis but are neither up-regulated nor down-regulated are depicted in white. Several canonical pathways were highlighted by IPA as being disrupted in FMDV-infected cells, including those involved in eIF2 signaling (20 out of a possible 222 molecules, P value = 3.35 × 10−18), CDK5 signaling (5 out of 94 molecules, P value = 2.97 × 10−4), mTOR signaling (6 out of a possible 210 molecules, P value = 1.69 × 10−3), integrin signaling (7 out of a possible 210 molecules, P value = 1.78 × 10−3), and mitochondrial dysfunction (5 out of a possible 175 molecules, P value = 3.31 × 10−3), etc. (Table 3 in the Supporting Information).

ratios of three representative proteins (β-tubulin, cathepsin D, and calpain-1) were in agreement with those obtained from SILAC approaches (Figure 8).

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DISCUSSION To the best of our knowledge, very few studies have been performed to analyze the interaction between foot-and-mouth disease virus and host cells using proteomic approaches. In the present work, to comprehensively screen such critical interplay and raise the detail of analysis that provides more biological information, we utilized high-throughput quantitative MS-based proteomics techniques, affording more opportunity to detect low abundance proteins as compared with traditional 2D gel electrophoresis.24 These data reveal novel whole-cell antigens that were affected by a porcine pathogen and therefore identify new candidate proteins, which may play either a direct or an indirect role in viral infection. Moreover, the Western blot results were consistent with the proteomic analysis. Under the condition of exquisite precision of SILAC coupled with MS quantitation, we are allowed to set the cutoff significance values to a B level ≤0.05, which reflected the high sensitivity in

Validation of Protein Identification and Quantification by Western Blotting

To confirm the differential expression of the proteomes identified, we analyzed selected proteins between infected and mock-infected cells through Western blotting. Although there are a limited number of appropriate immunological reagents for the quantified proteins in this study, the results showed that the 367



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Table 1. Proteins Regulated Significantly (B ≤ 0.05) in FMDV-Infected vs Mock-Infected IBRS-2 Cells, as Identified by LC− MS/MSa protein name

accession no.

ratios (infection/ control)

peptides

functions

proteins present in increased abundance in FMDV-infected cells oligosaccharyltransferase OST48 cystatin C

gi|10638222

gi| 110006616 ribosomal protein L4 gi| 112980817 60S ribosomal protein L6 gi| 113205608 60S ribosomal protein L10 gi| 113205616 protein phosphatase 1 catalytic subunit gamma isoform gi| 113205694 calpain small subunit 1 gi|115613 60S ribosomal protein L14 gi| 117661006 60S ribosomal protein L35 gi| 117661065 40S ribosomal protein S21 gi| 117661139 vascular cell adhesion molecule gi|1199461 integrin α V gi| 124517178 hexokinase-2 gi| 122134685 lysosomal trafficking regulator gi| 146741332 GNAS complex locus gi| 147223307 RAB22A, member RAS oncogene family gi| 147225162 karyopherin α 4 gi|15638988 retinol-binding protein 4 gi|3041715 fatty acid binding protein 5 gi| 166202107 ribosomal protein L19 gi|16798651 FK506 binding protein 7 gi| 194043960 interferon stimulated gene 15 gi| 182406743 solute carrier family 30 member 7 gi| 209553928 similar to U2 small nuclear ribonucleoprotein A gi| 194033769 similar to Huntingtin interacting protein K gi| 194034835 similar to ubiquitin-conjugating enzyme E2N gi| 194035345 similar to ribosomal protein L8 gi| 194035478 similar to methylosome protein 50 gi| 194036468 similar to ATP synthase, H+ transporting, mitochondrial F0 gi| complex, subunit B1 194036470 similar to uridine-cytidine kinase 2 gi| 194036831 similar to ADAM metallopeptidase with thrombospondin gi| type 1 motif, 4 194036878 similar to KAT protein gi| 194036918 similar to thioredoxin domain-containing protein 5 gi| precursor 194037976 similar to DEK oncogene isoform 1 gi| 194038067 similar to glutathione S-transferase A4 isoform 2 gi| 194038189

1.2295

9

1.4519

2

dolichyl-diphosphooligosaccharide-protein glycotransferase activity cysteine-type endopeptidase inhibitor activity

2.3021

4

structural constituent of ribosome

2.1886

7


1.409

4


1.717

12

hydrolase activity

1.2528 1.6684

10 3

calcium ion binding structural constituent of ribosome

2.0589

1


1.2864

3


1.758 1.4436

6 3

cell adhesion receptor activity, transforming growth factor beta binding

1.5731

5

hexokinase activity

2

endosome to lysosome transport

1.4207

8

signal transducer activity

1.6508

2

GTPase activity

1.9476 2.7884 1.2577

2 4 5

protein transporter activity transporter activity phosphatidylcholine biosynthetic process

1.4438 1.6278

2 1

structural constituent of ribosome protein folding

1.3062

2

type I interferon-mediated signaling pathway

1.886

1

zinc ion transmembrane transporter activity

4.9086

4

peptidase activity

1.4981

2

molecular function

1.2116

1

regulation of protein metabolic process

1.3003

6


1.2809

3

nuclear receptor transcription coactivator activity

1.3916

10

hydrogen ion transmembrane transporter activity

1.6079

3

phosphotransferase activity, alcohol group as acceptor

2.9822

1

metalloendopeptidase activity

1.3038

1

molecular function

1.4437

2

isomerase activity

1.2893

4

1.4516

1

regulation of transcription from RNA polymerase II promoter glutathione transferase activity

13.306

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Table 1. continued accession no.

protein name


peptides

functions

proteins present in increased abundance in FMDV-infected cells similar to ADP-dependent glucokinase similar to Golgin subfamily A member 4 similar to nuclear cap binding protein subunit 2, 20 kDa isoform 1 similar to SEC13-like 1 similar to ribosomal protein S24 isoform 2 similar to calcium-binding protein 39 (Protein Mo25) similar to potassium voltage-gated channel, subfamily H, member 7 fibronectin similar to 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase β-1 similar to sulfiredoxin 1 homologue RNA binding motif protein 39 isoform 1 similar to acyl-CoA thioesterase 9, partial similar to polymerase (DNA-directed), α, partial hypothetical protein similar to adenine nucleotide translocator 2 heme oxygenase 1 calpain-1 catalytic subugnit ribosomal protein L26-like 1 VLA-2 (integrin α2) A chain A, destrin, NMR, 20 structures S100C protein connective tissue growth factor 60S ribosomal protein L13a CCHC-type zinc finger, nucleic acid binding protein methionine adenosyltransferase II α ribonucleotide reductase M2 dipeptidyl peptidase IV succinate dehydrogenase cytochrome b560 subunit, mitochondrial transmembrane protein C9orf46 60S ribosomal protein L32 60S ribosomal protein L7 glutaminyl-tRNA synthetase intercellular adhesion molecule-1 FKBP1A-like cytochrome c oxidase subunit 4 isoform 1, mitochondrial 60S ribosomal protein L15 60S ribosomal protein L7a triosephosphate isomerase 1 ribosomal protein L18 P-glycoprotein class IA fibronectin 60S ribosomal protein L7

gi| 194038720 gi| 194040817 gi| 194041055 gi| 194041343 gi| 194042175 gi| 194043670 gi| 194044147 gi|55793113 gi| 194044250 gi| 194044425 gi| 194044531 gi| 194044820 gi| 194044830 gi| 194044838 gi| 194044922 gi|417137 gi|19883961 gi| 212378982 gi|2159 gi| 157829919 gi|217707 gi|2317892 gi|2500259 gi| 262072939 gi| 262204900 gi| 262263187 gi|28566188 gi| 290463150 gi| 298160936 gi|45268969 gi|45268971 gi|52631983 gi|55742638 gi|61098747 gi|6166026 gi|6174950 gi|6174957 gi|74275492 gi|89573899 gi|974545 gi|56608605 gi| 164023822

1.5702

1

phosphotransferase activity, alcohol group as acceptor

1.2591

4

golgi to plasma membrane protein transport

1.3626

2

nucleic acid binding

1.3303

1

1.2317

5

ATPase activity, coupled to transmembrane movement of ions, phosphorylative mechanism structural constituent of ribosome

1.2194

8

regulation of fatty acid oxidation

1.4593

3

transmembrane transport

2.141 1.4659

5 2

extracellular matrix protein, cell growth phospholipase C activity

1.6926

2

sulfiredoxin activity

1.3996

3

RNA processing

1.4658

1

molecular function

1.214

3

DNA-directed DNA polymerase activity

1.2106

1


1.2131

10

4.5362 1.5321 1.3319

2 2 4

heme oxygenase (decyclizing) activity calcium-dependent cysteine-type endopeptidase activity structural constituent of ribosome

1.4762 1.2485

2 7

identical protein binding positive regulation of actin filament depolymerization

1.2897 1.6639 1.5248 1.4478

2 1 2 3

calcium-dependent protein binding regulation of cell growth structural constituent of ribosome metal ion binding and nucleic acid binding

1.3867

5

methionine adenosyltransferase activity

1.7398

1

ribonucleoside-diphosphate reductase activity

1.4685 1.5429

3 1

serine-type peptidase activity electron carrier activity

1.4152

3

positive regulation of plasminogen activation

1.2215 2.8985 1.3232 1.5769 1.2231 1.254 1.5386 2.2584 2.4058 1.8615 1.4526 1.3098 1.673

6 5 7 1 6 5 1 5 12 1 1 11 8

369

adenine transmembrane transporter activity

structural constituent of ribosome structural constituent of ribosome proline-tRNA ligase activity cell−cell adhesion peptidyl-prolyl cis−trans isomerase activity cytochrome c oxidase activity structural constituent of ribosome structural constituent of ribosome triose-phosphate isomerase activity structural constituent of ribosome unknown extracellular matrix protein, cell growth structural constituent of ribosome



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Table 1. continued protein name

accession no.


peptides

functions

proteins present in increased abundance in FMDV-infected cells proteins present in decreased ATPase 8 gi|11055676 hematological and neurological expressed 1-like protein gi| 114326218 SNRPA gi| 115371743 polyprotein gi| 115418812 serine/threonine kinase 6 gi| 147223361 carnitine palmitoyltransferase I gi|14579225 tenascin XB gi| 147780437 IGF binding protein-2 gi|1491785 PHKG2 protein gi| 158144906 Na+, K+-ATPase gi|164380 complement cytolysis inhibitor gi|164409 Rho A gi|1695731 α-crystallin B chain gi|75063982 aminoacylase-I gi|4586438 general transcription factor IIIC, polypeptide 4, 90 kDa gi| 194033727 similar to WD repeat domain 5 gi| 194033744 similar to PC4 and SFRS1-interacting protein gi| 194034110 similar to CTAGE family, member 5 gi| 194034394 similar to WD repeat and HMG-box DNA-binding gi| protein 1 194034474 similar to LisH domain and HEAT repeat-containing gi| protein KIAA1468 194034593 similar to neural precursor cell expressed, developmentally gi| down-regulated gene 4-like, partial 194034610 similar to NADH dehydrogenase [ubiquinone]u gi| 1 β subcomplex subunit 9 194035601 similar to uncharacterized protein C8orf32 homologue gi| 194035608 similar to apoptotic chromatin condensation inducer in the gi| nucleus (Acinus) 194038901 similar to apolipoprotein D gi| 194041045 similar to microtubule-associated protein 4, partial gi| 194041183 similar to transducin-α gi| 194041214 similar to BCL2-associated athanogene 3 gi| 194042142 similar to glutamate dehydrogenase 1, partial gi| 194042272 similar to cadherin related 23 gi| 194042774 similar to ubiquitin fusion degradation protein 1 homologue gi| 194043450 similar to nucleoporin 35 kDa gi| 194043987 similar to eukaryotic translation initiation factor 2, gi| subunit 2 β, 38 kDa 194044484 similar to glutathione synthetase gi| 194044500 similar to ubiquitin-conjugating enzyme E2C gi| 194044685 cytochrome P450 gi| 197791176

abundance in FMDV-infected cells 0.39415 1 hydrogen ion transmembrane transporter activity 0.36594 3 unknown 0.62566

6

0.61897

15

0.42316

3

protein serine/threonine kinase activity

0.5441 0.42246

6 3

transferase activity signal transduction

0.074502 0.35358

1 2

regulation of cell growth phosphorylase kinase activity

0.64552 0.20465 0.38213 0.60133 0.40001 0.34499

5 2 2 8 3 2

0.26416

1

potassium ion transport positive regulation of protein ubiquitination small GTPase mediated signal transduction unfolded protein binding cellular amino acid metabolic process transcription initiation from RNA polymerase III promoter molecular function

0.44325

2

molecular function

0.36964

3

molecular function

0.13425

2

protein binding and DNA binding

0.20288

2

molecular function

0.47664

3

0.51618

3

negative regulation of sodium ion transport and protein modification process NADH dehydrogenase (ubiquinone) activity

0.38175

1

protein modification process

0.063783

2

apoptotic chromosome condensation

0.22992

2

transporter activity

0.57137

11

molecular function

0.41835

2

signal transducer activity

0.46022

9

antiapoptosis

0.47764

10

0.25587

3

calcium-dependent cell−cell adhesion

0.65146

8

molecular function

0.40159

1

molecular function

0.69125

9

translation initiation factor activity

0.2179

6

glutathione synthase activity

0.34762

2

regulation of protein metabolic process

0.48818

3

monooxygenase activity

370

RNA splicing RNA-dependent DNA replication

glutamate dehydrogenase [NAD(P)+] activity



Article

Table 1. continued protein name

accession no.


peptides

functions

proteins present in increased abundance in FMDV-infected cells proteins present in decreased gi| 206596732 30 kDa regulatory subunit of myosin phosphatase gi| 217039101 SFRS protein kinase 1 gi| 218140852 ubiquitin-conjugating enzyme E2L 3 gi| 219563058 A chain A, complex of bdellastasin with porcine trypsin gi| 257472074 ferrochelatase, mitochondrial gi| 281427372 calreticulin gi| 290756002 ran-specific GTPase-activating protein gi| 297307137 cytochrome c oxidase subunit VIa polypeptide 1 gi| 298160948 protein phosphatase-1 δ gi|3402903 TRK-fused protein gi|45269009 cell cycle exit and neuronal differentiation protein 1 gi|47523130 zinc finger protein 313 gi|47607447 cathepsin D gi| 122114359 claudin 1

a

abundance in FMDV-infected cells 0.55136 2 structural molecule activity and hydrolase activity 0.42513

2

catalytic activity

0.24791

1

protein kinase activity, regulation of mRNA processing

0.5669

3

0.12033

6

transcription coactivator and ubiquitin-protein ligase activity serine-type peptidase activity

0.55658

4

unknown

0.68039

14

0.64474

9

positive regulation of mitotic centrosome separation

0.50313

2

cytochrome c oxidase activity

0.65554 0.29636 0.44293 0.57323 0.69204

16 1 1 3 9

unfolded protein binding

phosphoprotein phosphatase activity unknown unknown cell differentiation aspartic-type endopeptidase activity

Please refer to Table 4 in the Supporting Information for detailed listings of peptides, PEP for peptide identification, etc.

Figure 4. Subcellular annotations of the proteins with differential expression (B ≤ 0.05) in IBRS-2 cells infected with FMDV. (A) Down-regulated proteins and (B) up-regulated proteins.

371



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Figure 5. Functional characterization of up-regulated and down-regulated proteins. (A) Diseases and disorders, (B) molecular and cellular functions, (C) physiological system development and functions, and (D) toxicity functions. More information is available in Table 3 in the Supporting Information. 372



Article

is associated with resistance to oxidative stress-induced apoptosis in different cell types, and reduced cathepsin D expression and activity lead to a decrease in reactive oxygen species (ROS).34 Intracellular levels of human thioredoxin are significantly elevated in transformed cells related to virus infection, including human T cell lymphotrophic virus-I (HTLV-I), Epstein−Barr virus (EBV), hepatitis B virus, and papilloma virus. Lymphocytes may be exposed to large amounts of oxidants because they migrate and function in inflammatory areas. Previous studies suggest that redox (reduction/ oxidation) status, strictly regulated by the buffering system such as thioredoxin, is closely related to several immune disorders as well as to viral infection.35 The major function of the cystatins is to protect the organism against endogenous proteases released from the lysosomes. All of the cystatins are competitive reversible, tight-binding inhibitors of cysteine proteases, which inhibit their targets in mM to sub-picomolar range.28 Cystatin C prevents herpes simplex virus replication and the induction of apoptosis in infected cells by decreasing cathepsin B.36 The overexpression of this protein also results in accumulation of fibronectin. Because proteins produced are responsible for cardiomyocyte injury and heart failure, this state may become important in studying the mechanism of viral myocarditis.37 Furthermore, cystatin C has immunoregulatory properties, taking part in the process of dendritic cells maturation and induction of tumor necrosis factor.38 Further large-scale studies are required to understand the roles and interrelation of cathepsin D and its target substrates in FMDVinfected cells. However, it is now clear that many types of viruses (including FMDV) are dependent on virus-coded or host cellular protease cleavages during replication. The cleavages are often observed during viral capsid assembly but may include additional processing reactions involved in viral nucleic acid metabolism. This mechanism makes specific antiproteases promising clinical candidates as viral inhibitors.39 In viruses, antipapain drug E64 or its lipophilic derivatives inhibited the FMDV Lpro, involved in the cleavage of P220, which shuts off host protein synthesis.40 Human rhinovirus 3C Protease, responsible for the cleavage of viral precursor polyproteins into structural and enzymatic proteins, was significantly inhibited by AG7088.41 In the host, cellular cystatin C enhanced antirotavirus action42 and distinctly reduced the yield of human coronaviruses.43 In poliovirus- and rhinovirusinfected cells, cystatin C may bind viral proteases to retain inhibitory activity and block viral replication.39 In this study, cystatin C was up-regulated approximately 1.4519-fold (Table 1). It will therefore be interesting to investigate whether cystatin C has constitutive antiviral activity in FMDV infection.

Figure 6. Overview of nine specific functional networks, each of which contained 10 or more “focus” proteins (proteins that were significantly up- or down-regulated), except network 9. Each box contains an arbitrary network number. The numbers between two networks represent the amount of overlapped proteins. More information is available in Table 3 in the Supporting Information.

measuring regulated protein spots. Overall, 77 proteins were found to be overexpressed, and 50 were down-regulated in IBRS-2 cells as a consequence of infection by FMDV. According to known cellular location of altered proteins, 19 kinds of ribosomal proteins, including components of both 40S and 60S ribosomal subunits, were up-regulated. It is suggested that overall overexpression of proteins distributed in ribosome may enhance the translation of viral genes and be required for propagation of positive-stranded RNA virus like FMDV in host cells.26 Cathepsin D and Its Target Substrates

Cysteine proteases are key components of lysosomal compartment, playing an important role in pathogen recognition and elimination, signal processing, and cell homeostasis in immune cells. The proper function of these proteases is regulated by dedicated inhibitors to reach a delicate balance. Pathogens can disrupt this balance by producing analogous proteases to subvert the host immune response.27 In the FMDV-infected cells, lysosomal cathepsin D is down-regulated, while cystatin C, one of the natural proteases inhibitors, is overexpressed. Cathepsin D is an aspartyl endopeptidase, participates in the process of MHC II-associated invariant chain, and generates suitable peptide epitopes to activate specific T cells, which is essential for normal functioning of the immune system.28 Viral dsRNA (double-stranded RNA) primarily recognized by the pattern-recognition receptors (PRRs) of the innate-immune system, result in activation of the inflammasome and apoptosis of infected cells through the secretion of mature cathepsin D.29 In addition, cathepsin D triggers activation of Bax, leading to selective release of AIF (apoptosis-inducing factor) from the mitochondria.30 Inhibition of cathepsin decreases the late phase of the apoptosis-associated killing of pneumococci in vitro.31 Taken together, reduction of this protein may indicate impairment of the immune system after FMDV infection and blockage of apoptosis. The former is consistent with the result of PRRSV (porcine reproductive and respiratory syndrome virus) infection.32 Cathepsin D could cleave several target substrates, which demonstrate numerous physiological and pathological functions based on the characteristic of protease.33 Here, thioredoxin, fibronectin, and cystatin c were up-regulated, all of which are a proteolytic target of cathepsin D. High thioredoxin expression

Integrin Signaling Pathway

Integrins are one of the important receptors for FMDV on the surface of susceptible cells, recognizing and binding to extracellular matrix (ECM) proteins to elicit signals that are transmitted into the cell. In this study, seven significantly regulated proteins were mapped to the integrin signaling pathway, including fibronectin 1 (FN1), calpain small subunit 1 (CAPNS1), Rho A (RHOA), calpain-1 (CAPN1), integrin α2 (ITGA2), integrin αv (ITGAV), and protein phosphatase-1 δ (PPP1CB) (Table 3 in the Supporting Information and Figure 7). Integrin expression is up-regulated during processes such as wound healing and inflammation.44 Furthermore, modulation 373



Article

Figure 7. IPA of proteins that were significantly altered in FMDV-infected IBRS-2 cells. Red, up-regulated proteins; green, significantly downregulated proteins; and white, proteins known to be in the network but that were not identified in our study. The color depth indicates the magnitude of the change in protein expression level. The shapes are indicative of the molecular class (i.e., protein family) (see Figure 1 in the Supporting Information for the legend). Lines connecting the molecules indicate molecular relationships. Dashed lines indicate indirect interactions, and solid lines indicate direct interactions. The arrow styles indicate specific molecular relationships and the directionality of the interaction.

of cellular integrin expression results in increased cell adhesion and virus yield in rotavirus infection45 and facilitates susceptibility of memory B cells to EBV infection.46 The higher expression level of integrin αv and integrin α2 in host

cells may be a critical factor in their greater susceptibility to FMDV infection, and this higher expression level provides a favorable environment for viral replication and pathogenesis. 374



Article

Together, the network complexes of these proteins in the integrin signaling shed light on the complicated interaction between each two of them and provide useful information to comprehend biological importance of those changes in the FMDV-infected cells. Physiological and Pathological Role of Other Changed Proteins

αB-Crystallin (CryAB) is the most abundant small heat shock protein (HSP) constitutively expressed in cardiomyocytes. It suppresses cardiac hypertrophic responses likely through attenuating NFAT signaling and protects cardiomyocytes from ischemia-induced necrosis in vitro and in vivo.53 Downregulation of CryAB after FMDV infection may trigger a severe form of desmin-related cardiomyopathy (DRCM), characterized by accumulation of misfolded proteins.54 It has been reported that an abnormal accumulation of CryAB with coarse granules was observed in severely affected cardiomyocytes in an FMDV-infected heart.55 The role that CryAB plays in the myocarditis associated with foot-and-mouth disease virus needs to be further investigated. Phospholipase Cβ1 (PLCB1) was up-regulated, and it may cause increased cell size and heightened expression of the hypertrophy-related marker gene, an atrial natriuretic peptide in the cardiomyocyte.56 Correspondingly, clusterin acts to prohibit apoptosis and to limit progression of autoimmune myocarditis, and it protects the heart from postinflammatory tissue destruction.57 In mice, myocarditis induced by Coxsackie virus B-3 infection was also associated with a clusterin response.58 Thus, the decrease in clusterin may be implicated in the tissue injury of FMDV infection. The altered expression of above proteins would be correlated with the highest risk of death from viral myocarditis in the young animals. To sum up, this integrative approach toward a proteomewide host−pathogen interaction analysis has revealed cellular factors that coordinately regulate the replication of FMDV and host response to infection. Some host proteins with respect to a particular function or disease phenotype, such as cystatin C, calpain, clusterin, and CryAB, are changed after FMDV infection. An understanding of their role in cells targeted by FMDV during infection will be critical for characterizing the contribution of these factors toward disease progression. This opens up new promising drug candidates for therapeutic intervention and a deeper understanding of viral pathogenesis.

Figure 8. Representative protein quantitative confirmation with immunoblot analysis. (A) β-Tubulin, (B) cathepsin D, and (C) calpain-1. SILAC ratios (H:L) are shown on the right side.

Infection of cells with FMDV leads to a CPE characterized by dramatic changes in cell shape and the production of numerous membrane vesicles within the cytoplasm. In contrast to early studies focused on membrane vesicles, more work has now been reported to reveal the effect of FMDV infection on the cytoskeleton.47 In our case, the functional significance of these changes in integrin and fibronectin expression is to initiate signaling events resulting in reorganization of the actin cytoskeleton and formation of focal complexes. Binding of these integrins to FN1 presumably involves postreceptor occupancy events such as cytoskeletal rearrangement and cell spreading that are crucial to immune function and virus infection.48 In addition, changes in other components of the cytoskeleton, and microtubules in particular, may play a key role in this process. α-, β-, and γ-Tubulin were not disrupted (Table 4 in the Supporting Information), indicating that a major contributor to the change in cell morphology is due to the dissociation of microtubules from the MTOC (microtubule organizing center), which was in accord with the result of BHK cells infected with FMDV. Cleavage of microtubule-associated protein4 (MAP4) (Table 1 and Table 4 in the Supporting Information) by 3Cpro of poliovirus could leave a truncated version to retain microtubule stability and allow vimentin to move into the virus replication complex complex.47 During early infection, human herpesvirus 8 induces the activation of the RhoA and, in doing so, modulates the acetylation of microtubules and promotes the trafficking of viral capsids and the establishment of infection, accompanied by the cytoskeletal rearrangements.49 Among the biochemical events that occur after these interactions, intracellular calcium ions are increased, and calpain is activated. Once activated, calpains selectively cleave a variety of substrates, many of which are membrane and cytoskeletal proteins. These results suggest that calpain may have an important role in cell adhesion and migration.50 In a previous study, calpains and cathepsins had been proposed to mediate neuronal cell death under a variety of neurotoxic conditions.51 In consideration of the multiple roles of calpain in the regulation of different cellular processes,52 a more profound endeavor would be focused on understanding its function in viral pathology. Changes in the intracellular milieu have been observed in response to viral infection due to alterations in the concentrations of important proteins. A decrease in PPP1CB involves both intergrins and various intracellular proteins that may regulate cytoskeletal organization. Because focal adhesion and stress-fiber formation are processes known to be dependent on the Rho-family of GTPases, calpain has been postulated to regulate these proteins, such as RhoA.50

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ASSOCIATED CONTENT

S Supporting Information *

Information for each significantly altered protein and unchanged proteins identified and data of MS/MS analyses of single peptide-based proteins with altered expression and functional characterization of up- and down-regulated proteins in FMDV-infected cells, networks, and pathways analysis. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: 0086-20-85280240. E-mail: [email protected] (M.L.). Tel/Fax: 0086-20-85280240. E-mail: [email protected]. cn (H.F.). Notes

The authors declare no competing financial interest. 375



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ACKNOWLEDGMENTS This work was supported by a grant from the National Program on Key Basic Research Project of China (2011CB504701), National High Technology Research and Development Program of China (2011AA10A209), Science & technology nova Program of Pearl River of Guangzhou (2012J2200086), and Public Industry (Agriculture) Specific Research Program (201303034).

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ABBREVIATIONS SILAC, stable isotope labeling with amino acids in cell culture; DMEM, Dulbecco's modified Eagle's medium; LC−MS/MS, liquid chromatography−mass spectrometry/mass spectrometry; IgG, immunoglobulin G; UTR, untranslated region; IFN-β, interferon β; NK cells, natural killer cells; HPLC, highperformance liquid chromatography; FDR, false discovery rate; PEP, posterior error probability; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; EBV, Epstein−Barr virus; HTLV-I, human T cell lymphotrophic virus-I

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Quantitative Proteomics by Amino Acid Labeling in Foot-and-Mouth

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