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

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Abstract
Table of Contents
Materials
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Literature Cited

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

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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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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 (H₂ O₂ ; 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
RANTES	RANTES	MAB678 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

^a Parts 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., ).

^b Santa Cruz Biotechnology, Inc.

^c R&D Systems.

^d ABBiotec (http://www.abbiotec.com/).

^e Lifespan 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., )

Basic Protocol 3: 3‐Nitrotyrosine Antibody Evaluation

Materials

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

Materials

Frozen plasma samples

Additional reagents and equipment for 3‐nitrotyrosine microarray ELISA ( protocol 1 )

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Figures

Figure 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.

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Figure 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.

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Figure 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.

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Figure 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.

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Videos

Literature Cited

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

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited