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
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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

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

  • 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 (H 2O 2; 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 7.5.1   Materials   Capture Antibodies for nTyr PTM ELISA Microarray a   Capture Antibodies for nTyr PTM ELISA Microarray

    Capture antibody Abbreviation Catalog no.
    Alpha‐lactalbumin ALB sc‐53151 b
    Angiotensinogen AGT MAB3156 c
    Aminopetidase N APN AF3815 c
    Amphiregulin AmR MAB262 c
    CD14 CD14 MAB3833 c
    Clusterin Clu AF2747 c
    Ceruloplasmin Cp sc‐69767 b
    C‐reactive protein CRP MAB17071 c
    Epidermal growth factor (EGF) EGF DY236 kit c
    EGF receptor (extracellular domain) EGFR AF‐231 c
    E‐selectin Esel AF‐724 c
    Basic fibroblast growth factor FGFb MAB233 c
    Fibrinogen Fibr ID6‐250310 d
    Heparin‐binding epidermal growth factor HBEGF AF‐292 c
    Hepatocyte growth factor HGF MAB694 c
    Intercellular adhesion molecular 1 ICAM MAB720 c
    Insulin‐like growth factor 1 IGF‐1 MAB291 c
    Lactoferrin LFn sc‐14431 b
    Leptin Leptin MAB398 c
    Galectin‐3BP/MAC‐2BP MAP‐2BP AF2226 c
    Matrix metalloprotease 1 MMP1 AF901 c
    Matrix metalloprotease 2 MMP2 AF902 c
    Matrix metalloprotease 9 MMP9 AF911 c
    Osteopontin OPN MAB14332 c
    Platelet‐derived growth factor AA PDGF MAB221 c
    Polymeric immunoglobulin receptor pIgR AF2717 c
    Lung surfactant protein A SP‐A LS‐C17957 e
    Transforming growth factor alpha TGFa AF‐239 c
    Tumor necrosis factor alpha TNFa MAB610 c
    u‐Plasminogen activator/urokinase uPR MAB1310 c
    Vascular endothelial growth factor VEGF AF‐293 c

     aParts of this table have been previously published. These parts are reproduced with permission from Journal of Proteome Research (Gonzalez et al., ) and Environmental Health Perspectives (Jin et al., ).
     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

  • 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., )

Basic Protocol 3: 3‐Nitrotyrosine Antibody Evaluation

  • Antigens needed to print and test (see Kato et al., , and van Dalen et al., , 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 ( protocol 1)

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

  • Frozen plasma samples
  • Additional reagents and equipment for 3‐nitrotyrosine microarray ELISA ( protocol 1)
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  •   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.
  •   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.
  •   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.
  •   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.


Literature Cited

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