High‐Throughput, Multiplexed Analysis of 3‐Nitrotyrosine in Individual Proteins

Hongjun Jin1, Richard C. Zangar1

1 Fundamental & Computational Sciences, Pacific Northwest National Laboratory, Richland, null
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 17.15
DOI:  10.1002/0471140856.tx1715s51
Online Posting Date:  February, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Reactive nitrogen species (RNS) and reactive oxygen species (ROS) are derived as a result of inflammation and oxidative stress and can result in protein modifications. As such, these protein modifications are used as biomarkers for inflammation and oxidative stress. In addition, modifications in single‐tissue‐associated proteins released into blood can provide insight into the tissue localization of the inflammation or oxidative stress. We have developed an enzyme‐linked immunosorbent assay antibody microarray platform to analyze the levels of 3‐nitrotyrosine in specific proteins in a variety of biological samples, including human plasma and sputum. Selective‐capture antibodies are used to immunoprecipitate individual proteins from samples onto isolated spots on the microarray chips. Then, a monoclonal antibody for 3‐nitrotyrosine is used to detect the amount of 3‐nitrotyrosine on each spot. Our studies suggest that this approach can be used to detect trace amounts of 3‐nitrotyrosine in human plasma and sputum. In this paper, we describe our antibody microarray protocol for detecting 3‐nitrotyrosine in specific proteins. Curr. Protoc. Toxicol. 51:17.15.1‐17.15.16. © 2012 by John Wiley & Sons, Inc.

Keywords: 3‐nitrotyrosine; ELISA microarray; multiplex; biomarker

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Production of ELISA Microarray Slides and Their Use in Assay for 3‐Nitrotyrosine
  • Basic Protocol 2: Generation of 3‐Nitrotyrosine Standards by Modification of BSA by Peroxynitrite
  • Basic Protocol 3: 3‐Nitrotyrosine Antibody Evaluation
  • Basic Protocol 4: Identifying a Useful Dilution of Human Plasma for Measuring 3‐Nitrotyrosine
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Production of ELISA Microarray Slides and Their Use in Assay for 3‐Nitrotyrosine

 Materials
  • Capture antibodies (Table 17.15.1)
  • Phosphate‐buffered saline (PBS; Fisher Scientific)
  • Alexa Fluor 546–conjugated goat anti—rabbit IgG (Invitrogen, cat. no. A‐11035),
  • Nonimmune (negative control) rabbit IgG
  • 2% and 0.1% (w/v) bovine serum albumin (BSA, IgG‐free, Jackson ImmunoResearch Laboratory, catalog number 001‐000‐162) in PBS
  • PBS‐T: 0.05% (v/v) Tween‐20 (Sigma, cat. no. P7949) in PBS
  • Desiccant
  • Plasma samples for analysis
  • Green fluorescent protein (GFP; Prospecbio, cat. no. PRO‐687; http://www.prospecbio.com/)
  • Biotinylated anti‐3‐nitrotyrosine monoclonal antibody (Hycult Biotechnology, cat. no. HM5002; http://www.hycultbiotech.com)
  • Biotinylated anti–green fluorescent protein (GFP) antibody (Rockland Immunochemicals, Inc, cat. no. 600‐401‐215)
  • Streptavidin‐conjugated horseradish peroxidase (HRP) (Jackson ImmunoResearch Laboratory, catalog number 016‐030‐084)
  • 1 µg/ml biotinylated tyramide (PerkinElmer)
  • 100 mM sodium borate buffer, pH 8.5 (see recipe)
  • Hydrogen peroxide (H2O2; Sigma, cat. no. H0904)
  • Streptavidin‐Cy3 (Jackson ImmunoResearch Laboratory, cat. no. 016‐160‐084)
  • Microarray printer (GeSiM Nanoplotter, http://www.gesim.de/)
  • 384‐well microtiter plates
  • Polylysine‐coated glass slides (Fisher Scientific, cat. no. 22‐037‐216), separated into 16 separate but identical wells
  • Super Pap Pen (Beckman Coulter; IM3580)
  • Airtight moisture chamber associated with Microarray Printer (GeSiM Nanoplotter, http://www.gesim.de/)
  • Dissecting microscope (optional)
  • Laser scanner: ScanArray Express HT laser scanner and data acquisition software (Perkin‐Elmer)
  • Slide rack (Pacific Southwest Lab Equipment Inc., cat. no. 4465; http://www.psl‐equip.com/)
  • Centrifuge (IEC Cetra‐MP4R with CAT‐244 slide adapter)
  • Vacuum desiccator and vacuum source
  • Vacuum sealer and bags
  • 96‐well assay plates (BD Falcon, cat. no. 353075)
  • Tightly sealable moisture container and lid (Pacific Southwest Lab Equipment Inc., http://www.psl‐equip.com/)
  • Oscillating mixer (Stovall Life Science, Inc., http://www.slscience.com/)
     
    Table 17.15.1 Capture Antibodies for nTyr PTM ELISA Microarraya
    Capture antibodyAbbreviationCatalog no.
    Alpha‐lactalbuminALBsc‐53151b
    AngiotensinogenAGTMAB3156c
    Aminopetidase NAPNAF3815c
    AmphiregulinAmRMAB262c
    CD14CD14MAB3833c
    ClusterinCluAF2747c
    CeruloplasminCpsc‐69767b
    C‐reactive proteinCRPMAB17071c
    Epidermal growth factor (EGF)EGFDY236 kitc
    EGF receptor (extracellular domain)EGFRAF‐231c
    E‐selectinEselAF‐724c
    Basic fibroblast growth factorFGFbMAB233c
    FibrinogenFibrID6‐250310d
    Heparin‐binding epidermal growth factorHBEGFAF‐292c
    Hepatocyte growth factorHGFMAB694c
    Intercellular adhesion molecular 1ICAMMAB720c
    Insulin‐like growth factor 1IGF‐1MAB291c
    LactoferrinLFnsc‐14431b
    LeptinLeptinMAB398c
    Galectin‐3BP/MAC‐2BPMAP‐2BPAF2226c
    Matrix metalloprotease 1MMP1AF901c
    Matrix metalloprotease 2MMP2AF902c
    Matrix metalloprotease 9MMP9AF911c
    OsteopontinOPNMAB14332c
    Platelet‐derived growth factor AAPDGFMAB221c
    Polymeric immunoglobulin receptorpIgRAF2717c
    RANTESRANTESMAB678c
    Lung surfactant protein ASP‐ALS‐C17957e
    Transforming growth factor alphaTGFaAF‐239c
    Tumor necrosis factor alphaTNFaMAB610c
    u‐Plasminogen activator/urokinaseuPRMAB1310c
    Vascular endothelial growth factorVEGFAF‐293c
     aParts of this table have been previously published. These parts are reproduced with permission from Journal of Proteome Research (Gonzalez et al., 2008a) and Environmental Health Perspectives (Jin et al., 2011).
     bSanta Cruz Biotechnology, Inc.
     cR&D Systems.
     dABBiotec (http://www.abbiotec.com/).
     eLifespan Biosciences (http://www.lsbio.com/).

Basic Protocol 2: Generation of 3‐Nitrotyrosine Standards by Modification of BSA by Peroxynitrite

 Materials
  • Fatty acid‐free BSA (Sigma, cat. no. A8860)
  • Phosphate‐buffered saline (PBS; Fisher Scientific)
  • Peroxynitrite stock solution (Millipore, cat. no. 20‐107)
  • 10 M NaOH
  • 12 M HCl
  • NanoDrop UV‐Vis ND‐2000 Spectrophotometer (Thermo Scientific)
  • 12‐ to 14‐kDa MWCO dialysis tubing (Spectrum Laboratories, Inc., cat. no. 132678)
  • Additional reagents and equipment for protein nitration (Beckman et al., 1994)

Basic Protocol 3: 3‐Nitrotyrosine Antibody Evaluation

 Materials
  • Antigens needed to print and test (see Kato et al., 2005, and van Dalen et al., 2009, for modification procedures; the only difference is the use of 4‐hydroxy‐3‐nitrobenzaldehyde to modify BSA):
    • KLH: keyhole limpet hemocyanin (Pierce, cat. no 77600): this antigen is a control for unmodified protein
    • OVA: ovalbumin (Pierce, cat. no. 77120): this antigen is a control for unmodified protein
    • NTO: 4‐hydroxy‐3‐nitrobenzaldehyde (Sigma‐Aldrich, cat. no. 55971)–labeled OVA: this antigen mimics 3‐nitrotyrosine modified OVA
    • BTK: 3‐bromo‐4‐hydroxybenzoic acid (Indofine Chemical Company, Inc., cat. no. 19‐155)–labeled KLH: this antigen mimics 3‐bromotyrosine‐modified KLH
    • BTO: 3‐bromo‐4‐hydroxybenzoic acid (Indofine Chemical Company, Inc., cat. no. 19‐155)–labeled OVA: this antigen mimics 3‐bromotyrosine modified OVA
    • CTO: 3‐chloro‐4‐hydroxybenzoic acid (Sigma‐Aldrich, cat. no. C44605)–labeled OVA: this antigen mimics 3‐chlorotyrosine–modified OVA
    • BSA‐nTyr: peroxynitritetreated BSA: this antigen is a positive control for 3‐nitrotyrosine
    • BSA‐BrO: sodium hypobromite (Fisher Scientific, ca. no. NC9754116)–treated BSA: this antigen is a control for bromated protein
  • Phosphate‐buffered saline (PBS; Fisher Scientific)
  • PBS‐T: 0.05% (v/v) Tween‐20 (Sigma, cat. no. P7949) in PBS
  • 1% (w/v) bovine serum albumin (BSA) in PBS‐T
  • 3‐nitrotyrosine antibodies used for detection antibodies:
    • Antibody 1: goat anti‐nTyr, polyclonal (Abcam, cat. no. Ab27648)
    • Antibody 2: goat anti‐nTyr, polyclonal (Meridian Life Science, cat. no. K97520G; http://meridianlifescience.com/)
    • Antibody 3: rabbit anti‐nTyr, polyclonal (Molecular Probes, cat. no. A‐21285)
    • Antibody 4: mouse monoclonal antibody to nTyr (monoclonal strain HM11; Thermo Scientific, cat. no. MA1‐35729 or Hycult Biotechnology, cat. no. HM5001; http://www.hycultbiotech.com/)
  • Secondary antibodies (depending on source of primary antibody):
    • Mouse anti‐rabbit IgG (Jackson ImmunoResearch Laboratory, cat. no. 211‐035‐109)
    • HRP‐conjugated rabbit anti‐goat IgG (Jackson ImmunoResearch Laboratory, cat. no. 305‐035‐003)
    • HRP‐conjugated goat anti‐mouse IgG (Jackson ImmunoResearch Laboratory, cat. no. 115‐035‐003)
  • Additional reagents and equipment for printing array slides (Basic Protocol 1)

Basic Protocol 4: Identifying a Useful Dilution of Human Plasma for Measuring 3‐Nitrotyrosine

 Materials
  • Frozen plasma samples
  • Additional reagents and equipment for 3‐nitrotyrosine microarray ELISA (Basic Protocol 1)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •  FigureFigure 17.15.1 Comparison of 3‐nitrotyrosine ELISA microarray procedure with typical multiplex sandwich ELISA microarray analysis. In the standard, multiplexed ELISA microarray analysis, the various detection antibodies are combined into a single solution (left column). In the 3‐nitrotyrosine (PTM) ELISA microarray, a single detection antibody for 3‐nitrotyrosine is used (right column). Based on the fluorescence intensity in the array spots, the relative amount of 3‐nitrotyrosine modifications is estimated.
    View Image
  •  FigureFigure 17.15.2 ELISA microarray slide and chip format. The arrows and circles identify four replicate assays that, like all assays, are printed once in each quadrant in each well of the 16‐well glass slide.
    View Image
  •  FigureFigure 17.15.3 Evaluation of 3‐nitrotyrosine antibodies for assay sensitivity and specificity. (A) Diagram of antigens printed on the slides. (B, C, and D) Three different antibodies reacted with printed antigens. The antibody in panel D is selected for the best specificity and sensitivity to 3‐nitrotyrosine modification. Key: KLH: keyhole limpet hemocyanin; OVA: Ovalbumin; NTO: 3‐nitro‐4‐hydroxybenzoic acid–labeled OVA. This antigen mimics 3‐nitrotyrosine–modified OVA; BTK: 3‐bromo‐4‐hydroxybenzoic acid–labeled KLH. This antigen mimics 3‐bromotyrosine modified KLH; BTO: 3‐bromo‐4‐hydroxybenzoic acid–labeled OVA. This antigen mimics 3‐bromotyrosine modified OVA; CTO: 3‐choloro‐4‐hydroxybenzoic acid–labeled OVA. This antigen mimics 3‐chlorotyrosine modified OVA. BSA‐nTyr: peroxynitrite‐treated BSA. This antigen is a positive control for 3‐nitrotyrosine; BSA‐BrO: sodium hypobromite–treated BSA.
    View Image
  •  FigureFigure 17.15.4 Defining a useful dilution of human plasma for measuring 3‐nitrotyrosine. ELISA microarray results for the dilution of human plasma. Four samples were diluted with 0.1% BSA/PBS at 2‐, 5‐, 10‐, 20‐, 100‐, 1000‐, 5000‐, and 25000‐fold. The fluorescence intensity of 3‐nitrotyrosine from each diluted assay was plotted with folds of dilution (X‐dimension label). The different capture antibodies are color coded in the inset key. The results suggest that the 3‐nitrotyrosine level is saturated across all measured samples when plasma samples are 20‐fold or less diluted.
    View Image

Videos

Literature Cited

Literature Cited
    Beckman, J.S., Chen, J., Ischiropoulos, H., and Crow, J.P. 1994. Oxidative chemistry of peroxynitrite. Methods Enzymol. 233: 229‐240.
    Daly, D.S., Anderson, K.K., White, A.M., Gonzalez, R.M., Varnum, S.M., and Zangar, R.C. 2008. Predicting protein concentrations with ELISA microarray assays, monotonic splines and Monte Carlo simulation. Stat. Appl. Genet. Mol. Biol. 7: Article21.
    Daly, D.S., Anderson, K.K., Seurynck‐Servoss, S.L., Gonzalez, R.M., White, A.M., and Zangar, R.C. 2010. An internal calibration method for protein‐array studies. Stat. Appl. Genet. Mol. Biol. 9: Article 14.
    Gonzalez, R.M., Seurynck‐Servoss, S.L., Crowley, S.A., Brown, M., Omenn, G.S., Hayes, D.F., and Zangar, R.C. 2008a. Development and validation of sandwich ELISA microarrays with minimal assay interference. J. Proteome Res. 7:2406‐2414.
    Gonzalez, R.M., Varnum, S.M., and Zangar, R.C. 2008b. "Sandwich ELISA microarrays: Generating reliable and reproducible assays for high‐throughput screens". In Biomarker Methods in Drug Discovery and Development (Methods in Pharmacology and Toxicology), 1st ed. ( F. Wang, ed.) pp. 273‐290. Humana Press, Totowa, New Jersey.
    Gow, A.J., Thom, S.R., and Ischiropoulos, H. 1998. Nitric oxide and peroxynitrite‐mediated pulmonary cell death. Am. J. Physiol. 274:L112‐L118.
    Ischiropoulos, H. 1998a. Biological tyrosine nitration: A pathophysiological function of nitric oxide and reactive oxygen species. Arch. Biochem. Biophys. 356:1‐11.
    Ischiropoulos, H. 1998b. Living and dying with reactive species. Focus on “peroxynitrite induces apoptosis of HL‐60 cells by activation of a caspase‐3 family protease”. Am. J. Physiol. 274:C853‐C854.
    Jin, H. and Zangar, R.C. 2009. Protein modifications as potential biomarkers in breast cancer. Biomark Insights 4:191‐200.
    Jin, H. and Zangar, R.C. 2010. Antibody microarrays for high‐throughput, multianalyte analysis. Cancer Biomark 6:281‐290.
    Jin, H., Webb‐Robertson, B.J., Peterson, E.S., Tan, R., Bigelow, D.J., Scholand, M.B., Hoidal, J.R., Pounds, J.G., and Zangar, R.C. 2011. Smoking, COPD and 3‐nitrotyrosine levels of plasma proteins. Environ. Health Perspect. 119:1314‐1320.
    Kato, Y., Kawai, Y., Morinaga, H., Kondo, H., Dozaki, N., Kitamoto, N., and Osawa, T. 2005. Immunogenicity of a brominated protein and successive establishment of a monoclonal antibody to dihalogenated tyrosine. Free Radic. Biol. Med. 38:24‐31.
    Kodama, N., Kambayashi, Y., Kubo, M., Nobukuni, Y., Kimura, S., Nakamura, H., and Ogino, K. 2004. Induction of myeloperoxidase and nitrotyrosine formation in a human eosinophilic leukemia cell line, EoL‐1. Cell Biochem. Funct. 22:105‐112.
    Mohiuddin, I., Chai, H., Lin, P.H., Lumsden, A.B., Yao, Q., and Chen, C. 2006. Nitrotyrosine and chlorotyrosine: Clinical significance and biological functions in the vascular system. J. Surg. Res. 133:143‐149.
    Pehar, M., Cassina, P., Vargas, M.R., Castellanos, R., Viera, L., Beckman, J.S., Estevez, A.G., and Barbeito, L. 2004. Astrocytic production of nerve growth factor in motor neuron apoptosis: Implications for amyotrophic lateral sclerosis. J. Neurochem. 89:464‐473.
    Peluffo, G. and Radi, R. 2007. Biochemistry of protein tyrosine nitration in cardiovascular pathology. Cardiovasc. Res. 75:291‐302.
    Seurynck‐Servoss, S.L., White, A.M., Baird, C.L., Rodland, K.D., and Zangar, R.C. 2007. Evaluation of surface chemistries for antibody microarrays. Anal. Biochem. 371: 105‐115.
    van Dalen, C.J., Aldridge, R.E., Chan, T., Senthilmohan, R., Hancox, R.J., Cowan, J.O., Taylor, D.R., Town, G.I., and Kettle, A.J. 2009. Bromotyrosines in sputum proteins and treatment effects of terbutaline and budesonide in asthma. Ann. Allergy Asthma Immunol. 103:348‐353.
    Varnum, S.M., Woodbury, R.L., and Zangar, R.C. 2004. A protein microarray ELISA for screening biological fluids. Methods Mol. Biol. 264: 161‐172.
    White, A.M., Daly, D.S., Varnum, S.M., Anderson, K.K., Bollinger, N., and Zangar, R.C. 2006. ProMAT: Protein microarray analysis tool. Bioinformatics 22: 1278‐1279.
    White, A.M., Daly, D.S., and Zangar, R.C. 2011. Analysis of high‐throughput ELISA microarray data. Methods Mol. Biol. 694:191‐211.
    Woodbury, R.L., Varnum, S.M., and Zangar, R.C. 2002. Elevated HGF levels in sera from breast cancer patients detected using a protein microarray ELISA. J. Proteome Res. 1: 233‐237.
    Zangar, R.C., Varnum, S.M., Covington, C.Y., and Smith, R.D. 2004. A rational approach for discovering and validating cancer markers in very small samples using mass spectrometry and ELISA microarrays. Dis. Markers 20:135‐148.
    Zangar, R.C., Daly, D.S., and White, A.M. 2006. ELISA microarray technology as a high‐throughput system for cancer biomarker validation. Expert Rev. Proteomics 3:37‐44.
    Zangar, R.C., Daly, D.S., White, A.M., Servoss, S.L., Tan, R.M., and Collett, J.R. 2009. ProMAT calibrator: A tool for reducing experimental bias in antibody microarrays. J. Proteome Res. 8:3937‐3943.
    Zangar, R.C., Jin, H., Hallstrand, T.S., Bigelow, D.J., and Pounds, J.G. 2011. Halotyrosine modifications in sputum proteins are indicative of asthma severity. Am. J. Respir. Crit. Care Med. 183:A4389.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library