Enrichment and Detection of Tyrosine‐Nitrated Proteins

Frank Dekker1, Nicolas Abello2, Rosalina Wisastra1, Rainer Bischoff2

1 Pharmaceutical Gene Modulation, Groningen Research Institute of Pharmacy, University of Groningen, The Netherlands, 2 Analytical Biochemistry, Groningen Research Institute of Pharmacy, University of Groningen, The Netherlands
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 14.13
DOI:  10.1002/0471140864.ps1413s69
Online Posting Date:  August, 2012
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Abstract

Nitrotyrosine is a post‐translationally modified amino acid with distinctly different properties than tyrosine or any other of the genetically encoded amino acids. Detecting proteins containing nitrotyrosine is the first step towards a better understanding of the role of nitrotyrosine in health and disease. Moreover, quantifying the extent of nitrotyrosine and determining its location in a protein forms the basis for a better understanding of the effect of tyrosine nitration on biological function. Described in this unit is a method to detect tyrosine‐nitrated proteins in tissue sections and on western blots after creating a fluorescent complex between aminotyrosine, salicylaldehyde, and Al3+. In addition, an approach is detailed for labeling aminotyrosine with biotin to enrich peptides from complex samples. Both methods require reduction of nitrotyrosine to aminotyrosine, which can be achieved with sodium dithionite or hemin plus dithiothreitol. Curr. Protoc. Protein Sci. 69:14.13.1‐14.13.19. © 2012 by John Wiley & Sons, Inc.

Keywords: nitrotyrosine; nitration; fluorescence; mass spectrometry; tissue analysis; oxidative stress

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Fluorescent Staining of Protein‐Bound Nitrotyrosine on Immunoblots
  • Alternate Protocol 1: Immunostaining of Protein‐Bound Nitrotyrosine on Immunoblots
  • Basic Protocol 2: Visualization of Nitrotyrosine in Tissue Sections by Fluorescent Staining
  • Basic Protocol 3: Chemical Labeling and Enrichment of Nitrotyrosine‐Containing Peptides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Fluorescent Staining of Protein‐Bound Nitrotyrosine on Immunoblots

  Materials
  • 3‐Nitro‐4‐hydroxyphenylacetic acid (NHPA; Aldrich)
  • Acetonitrile, HPLC‐S gradient grade (BioSolve)
  • Triethylamine (C 6H 15N)
  • Ethyl chloroformate (C 3H 5ClO 2)
  • Bovine serum albumin (BSA, Fraction V powder, ≥96%, Sigma)
  • 0.1 M carbonate buffer, pH 9.6 (see recipe)
  • 0.1 M phosphate buffer: 12 g/liter anhydrous monosodium phosphate, pH 7.5 with NaOH
  • Tris buffer: 0.1 M Tris‐Cl, pH 7.5 with NaOH
  • 2‐Nitrophenol (HOC 6H 4NO 2)
  • Dimethyl sulfoxide (DMSO)
  • Nitrotyrosine samples
  • SDS sample buffer (see recipe)
  • 1× SDS running buffer (see recipe)
  • Blotting buffer (see recipe)
  • Reducing solution (see recipe)
  • Staining solution (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • Rocking plate (e.g., Bibby Stuart, Platform Rocker STR 6)
  • Slide‐A‐Lyzer with 10‐kDa cutoff (ThermoScientific)
  • 500‐ml glass beaker
  • 96‐well plates (Costar)
  • Absorbance microplate reader (e.g., ThermoMax, Molecular Devices)
  • 110°C heating block
  • Tissue paper
  • Blot scanner (e.g., Fuji Film LAS‐4000)
  • Image reader (e.g., Image Reader LAS‐4000 v. 2.0)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1) and immunoblotting (unit 10.7)

Alternate Protocol 1: Immunostaining of Protein‐Bound Nitrotyrosine on Immunoblots

  • Blood plasma samples collected in EDTA‐containing (purple) Vacutainer tubes
  • Anti‐nitrotyrosine‐agarose beads: mouse monoclonal IgG anti‐nitrotyrosine, clone 1A6, agarose conjugate (Upstate‐Millipore, cat. no. 16‐163)
  • DPBS: 1× Dulbecco's phosphate‐buffered saline, diluted in dH 2O from 10× stock (GIBCO‐Invitrogen)
  • Blocking solution A: 5% (w/v) fat‐free milk powder in DPBS, prepare fresh
  • DPBST: DPBS containing 0.05% (v/v) Tween 20 (Sigma‐Aldrich)
  • Primary antibody: rabbit polyclonal IgG anti‐nitrotyrosine (Upstate‐Millipore, cat. no. 06‐284)
  • Secondary antibody: horseradish peroxidase (HRP)−conjugated swine anti‐rabbit (Dako Cytomation, cat. no. P021702)
  • Enhanced chemiluminescence (ECL) solutions (GE Healthcare)
  • Gel imaging system (e.g., G:BOX Chemi XT, Fujifilm, Japan)
  • Additional reagents and equipment for Bradford protein assays (unit 3.4)

Basic Protocol 2: Visualization of Nitrotyrosine in Tissue Sections by Fluorescent Staining

  Materials
  • Fresh frozen tissue sections cut with a cryostat (4 µm), slide‐mounted
  • Acetone
  • DPBS: 1× Dulbecco's phosphate‐buffered saline, diluted in dH 2O from 10× stock (GIBCO‐Invitrogen)
  • Blocking solution B: 1% (w/v) bovine serum albumin in DPBS, prepare fresh
  • Primary antibody: rabbit polyclonal IgG anti‐nitrotyrosine (Upstate‐Millipore, cat. no. 06‐284)
  • DPBST: DPBS containing 0.05% (v/v) Tween 20 (Sigma‐Aldrich)
  • Secondary antibody: FITC‐conjugated goat anti‐rabbit (Abcam, cat. no. AB6717)
  • AF1 anti‐bleaching agent (glycerol/PBS; Citifluor)
  • Reducing solution (see recipe)
  • Staining solution (see recipe)
  • Hair blower (dryer)
  • Hydrophobic barrier pen (e.g., ImmEdge, Vector Laboratories)
  • 24 × 50−mm cover slips (Menzel‐Gläser)
  • Confocal microscope
  • Fluorescence microscope (e.g., Leica DAS Microscope LEITZ DMR) with filter A (UV, 340‐380 nm ex., 425 nm em.), filter +L5 (blue, 480/40 nm ex., 527/30 nm em.), and filter +N3 (green, 546/12 nm ex., 600/40 nm em.)
  • Digital camera (e.g., Leica DC350FX with Leica‐QWin software)

Basic Protocol 3: Chemical Labeling and Enrichment of Nitrotyrosine‐Containing Peptides

  Materials
  • 500 µM human angiotensin II (DRVYIHPF) acetate (Sigma) in water
  • Sodium phosphate buffer (see recipe): 500 mM (pH 7.5), 100 mM (pH 8.0), and 250 mM (pH 8.0)
  • Peroxynitrite solution (160‐200 mM in 4.7% NaOH; Calbiochem)
  • Methanol (BioSolve)
  • Nitrogen gas
  • Protein samples of interest
  • Trypsin (Sigma, cat. no. T1426)
  • 500 mM acetic acid N‐hydroxysuccinimide ester (NHS‐acetate, MP Biomedicals) in dimethyl sulfoxide (DMSO; Merck KGaA)
  • 440 mM dithiothreitol (DTT; Sigma) in 250 mM sodium phosphate buffer, pH 8.0
  • 550 µM hemin (Sigma‐Fluka) in 250 mM sodium phosphate buffer, pH 8.0
  • 115 mM biotinyl N‐hydroxysuccinimide ester (NHS‐biotin; Sigma) in DMSO
  • 1% and 0.425‰ (v/v; 11 mM) formic acid (Merck KGaA)
  • SCX elution buffer (see recipe)
  • Avidin‐agarose beads: 50% slurry of monomeric avidin immobilized on agarose beads (Pierce, Perbio Science)
  • DPBS: 1× Dulbecco's phosphate‐buffered saline, diluted in dH 2O from 10× stock (GIBCO‐Invitrogen)
  • 8 M guanidinium‐HCl, pH 1.4
  • Oasis HLB column (Waters), preconditioned with 1 ml methanol and 1 ml water
  • Strong cation‐exchange cartridge (3‐ml LiChrolut SCX; Merck), preconditioned with 3 ml methanol and 3 ml of 11 mM formic acid
  • Vacuum centrifuge (Concentrator 5301, Eppendorf)
  • Rotary shaker
  • Gel loader tips
NOTE: All incubations at 25° and 37°C are performed in a Thermomixer (Eppendorf) at 450 rpm.
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Figures

Videos

Literature Cited

Literature Cited
   Abello, N., Kerstjens, H.A., Postma, D.S., and Bischoff, R. 2007. Selective acylation of primary amines in peptides and proteins. J. Proteome Res. 6:4770–4776.
   Abello, N., Kerstjens, H.A., Postma, D.S., and Bischoff, R. 2009. Protein tyrosine nitration: Selectivity, physicochemical and biological consequences, denitration and proteomics methods for the identification of tyrosine‐nitrated proteins. J. Proteome Res. 8:3222‐3238.
   Abello, N., Barroso, B., Kerstjens, H.A., Postma, D.S., and Bischoff, R. 2010. Chemical labeling and enrichment of nitrotyrosine‐containing peptides. Talanta 80:1503‐1512.
   Beal, M.F. 2002. Oxidatively modified proteins in aging and disease. Free Rad. Biol. Med. 32:797‐803.
   Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72:248‐254.
   Drăguşanu, M., Petre, B.A., and Przybylski, M. 2011. Epitope motif of an anti‐nitrotyrosine antibody specific for tyrosine‐nitrated peptides revealed by a combination of affinity approaches and mass spectrometry. J. Peptide Sci. 17:184‐191.
   Dijkstra, G., Moshage, H., van Dullemen, H.M., de Jager‐Krikken, A., Tiebosch, A.T., Kleibeuker, J.H., Jansen, P.L., and van Goor, H. 1998. Expression of nitric oxide synthases and formation of nitrotyrosine and reactive oxygen species in inflammatory bowel disease. J. Pathol. 186:416‐421.
   Haddad, I.Y., Pataki, G., Hu, P., Galliani, C., Beckman, J.S., and Matalon, S. 1994. Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury. J. Clin. Invest. 94:2407‐2413.
   Hanazawa, T., Kharitonov, S.A., and Barnes, P.J. 2000. Increased nitrotyrosine in exhaled breath condensate of patients with asthma. Am. J. Respir. Crit. Care Med. 162:1273‐1276.
   Mapp, P.I., Klocke, R., Walsh, D.A., Chana, J.K., Stevens, C.R., Gallagher, P.J., and Blake, D.R. 2001. Localization of 3‐nitrotyrosine to rheumatoid and normal synovium. Arthritis Rheum. 44:1534‐1539.
   Ohshima, H., Friesen, M., Brouet, I., and Bartsch, H. 1990. Nitrotyrosine as a new marker for endogenous nitrosation and nitration of proteins. Food Chem. Toxicol. 28:647‐652.
   Vattemi, G., Mechref, Y., Marini, M., Tonin, P., Minuz, P., Grigoli, L., Guglielmi, V., Klouckova, I., Chiamulera, C., Meneguzzi, A., Di Choi, M., Tedesco, V., Lovato, L., Degan, M., Arcaro, G., Lechi, A., Novotny, M.V., and Tomelleri, G. 2011. Increased protein nitration in mitochondrial diseases: Evidence for vessel wall involvement. Mol. Cell Proteomics epub 10:M110.002964.
   Wisastra, R., Poelstra, K., Bischoff, R., Maarsingh, H., Haisma, H.J., and Dekker, F.J. 2011. Antibody‐free detection of protein tyrosine nitration in tissue sections. Chembiochem 12:2016‐2020.
   Zhan, X. and Desiderio, D.M. 2009. Mass spectrometric identification of in vivo nitrotyrosine sites in the human pituitary tumor proteome. Methods Mol. Biol. 566:137‐163.
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