Identification and Characterization of Oxylipid‐Protein and Peptide Conjugates by Mass Spectrometry

Woon‐Gye Chung1, Claudia S. Maier1

1 Oregon State University, Department of Chemistry, Corvallis, Oregon
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 17.9
DOI:  10.1002/0471140856.tx1709s35
Online Posting Date:  February, 2008
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Abstract

The modification of proteins by reactive products of lipid peroxidation is associated with a large number of diseases and biological aging; thus, methods that enable the characterization of oxylipid‐protein and/or peptide conjugates are highly in demand. This unit outlines a chemical labeling approach to identifying and characterizing proteins modified by lipid peroxidation products. It also outlines two approaches for mass spectrometry–based identification and detailed characterization of oxylipid conjugates. The first combines chemical labeling of oxylipid‐protein conjugates using an aldehyde‐specific biotinylation reagent, electrophoretic separation, and mass spectrometry–based identification of the biotinylated proteins. In the second approach, protein extracts are treated with the aldehyde‐specific reagent, proteolyzed using trypsin, and the biotinylated peptides are enriched using immobilized monomeric avidin. The enriched peptide fractions are submitted to tandem mass spectrometry for determining the peptide sequence information, site of the modification, and chemical nature of the oxylipid. Curr. Protoc. Toxicol. 35:17.9.1‐17.9.19. © 2008 by John Wiley & Sons, Inc.

Keywords: lipid peroxidation; oxylipid; chemical labeling; mass spectrometry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Chemical Labeling of Oxylipid‐Protein Conjugates with Aldehyde‐Reactive Probe (ARP) and Separation by One‐Dimensional SDS‐PAGE
  • Alternate Protocol 1: Separate ARP‐Treated Protein Samples by Two‐Dimensional SDS‐PAGE
  • Basic Protocol 2: Detection of ARP‐Labeled Proteins on Blots Using Avidin‐Affinity Staining
  • Basic Protocol 3: Labeling Protein Extracts with ARP in Combination with Avidin‐Affinity Enrichment
  • Support Protocol 1: In‐Gel Trypsin Digestion of Samples
  • Support Protocol 2: Desalting Tryptic Peptide Mixtures Using C18 ZipTips
  • Support Protocol 3: Sample Preparation for MALDI MS Analysis Using a Nano‐HPLC Equipped with Target Spotter
  • Basic Protocol 4: Mass Spectrometry and Data Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Chemical Labeling of Oxylipid‐Protein Conjugates with Aldehyde‐Reactive Probe (ARP) and Separation by One‐Dimensional SDS‐PAGE

  Materials
  • 50 to 200 µg protein sample (homogenate or lysate)
  • 10 mM sodium phosphate buffer, pH 7.4 ( appendix 2A)
  • 30 mM aldehyde reactive probe (ARP): 30 mM N′‐aminooxymethylcarbonylhydrazine‐D‐biotin (Dojindo Laboratories)/10 mM sodium phosphate buffer, pH 7.4 (see appendix 2A); store up to 6 months at −80°C
  • Laemmli sample buffer (Bio‐Rad)
  • 2‐mercaptoethanol (Bio‐Rad)
  • Biotinylated SDS‐PAGE molecular mass standards (Bio‐Rad)
  • Polyacrylamide gel (see appendix 3F): percent acrylamide appropriate for size of proteins to be separated
  • Running buffer: 25 mM Tris base/192 mM glycine/0.1% (w/v) SDS, pH 8.3
  • Bio‐Safe Coomassie stain (Bio‐Rad)
  • 95°C heating block
  • Additional reagents and equipment for separating proteins by SDS‐PAGE ( appendix 3F)

Alternate Protocol 1: Separate ARP‐Treated Protein Samples by Two‐Dimensional SDS‐PAGE

  Materials
  • Rehydration buffer (see recipe)
  • 50 to 200 µg ARP‐treated protein sample (see protocol 1, steps 1 to 3)
  • IPG strip, pH 3 to 10 (Bio‐Rad)
  • Mineral oil
  • Reducing equilibration buffer (see recipe)
  • Alkylating equilibration buffer (see recipe)
  • SDS‐PAGE running buffer (see recipe)
  • 13.5 × 8−cm 8% to 16% gradient polyacrylamide gel (see appendix 3F)
  • Overlay agarose (see recipe)
  • Biotinylated molecular mass standard (Bio‐Rad)
  • 10% (v/v) methanol/7% (v/v) acetic acid
  • SYPRO Ruby or IEF gel staining solution (Bio‐Rad)
  • Bath sonicator, optional
  • Protean isoelectric focusing (IEF) cell (e.g., Bio‐Rad)
  • Paper wick
  • Forceps
  • Rehydration tray (plastic tray of appropriate size)
  • Fluorescence scanner (for SYPRO Ruby stain; Molecular Imager FX, Bio‐Rad) or densitometer and documentation system (for IEF gel stain; e.g., Kodak Image Station 440 CF)
  • Additional reagents and equipment for separating proteins by SDS‐PAGE ( appendix 3F)

Basic Protocol 2: Detection of ARP‐Labeled Proteins on Blots Using Avidin‐Affinity Staining

  Materials
  • Methanol
  • Transfer buffer (see recipe)
  • One‐dimensional polyacrylamide gel with 20 µg separated ARP‐treated protein ( protocol 1) or two‐dimensional polyacrylamide gel with 50 to 200 µg separated ARP‐treated protein ( protocol 2), both with 0.26 µg biotinylated SDS‐PAGE molecular weight standard
  • Tris‐buffered saline (TBS; appendix 2A) containing 0.5% (v/v) Tween‐20 (TBS‐T5) and 0.1% (v/v) Tween‐20 (TBS‐T1)
  • 1 mg/ml NeutrAvidin horseradish peroxide conjugate (Pierce) stock solution: reconstituted with 0.4 ml H 2O and diluted to 2 ml with PBS ( appendix 2A); store up to 2 years at −20°C
  • SuperSignal West Pico chemiluminescent substrate (Pierce)
  • Blotting membrane: PVDF‐P (Millipore) or nitrocellulose (Bio‐Rad)
  • Blotting apparatus with cassette and sponge pads
  • Blotter filter papers (Bio‐Rad)
  • Test tube (for rolling out air bubbles from transfer stack)
  • Magnetic stirrer
  • Additional reagents and equipment for exposing the membrane to the X‐ray film, and developing, scanning, and quantifying the detected spots ( appendix 3D)

Basic Protocol 3: Labeling Protein Extracts with ARP in Combination with Avidin‐Affinity Enrichment

  Materials
  • 400 to 600 µg protein sample
  • 10 mM sodium phosphate buffer, pH 7.4 (see appendix 2A)
  • 30 mM aldehyde reactive probe (ARP): 30 mM N′‐ aminooxymethylcarbonylhydrazine‐D‐biotin (Dojindo Laboratories)/10 mM sodium phosphate buffer, pH 7.4 ( appendix 2A); store up to 6 months at −80°C
  • Trypsin, sequencing‐grade modified (Promega)
  • 1% (v/v) acetic acid
  • Milli‐Q‐purified H 2O (Millipore)
  • 50% slurry of Ultralink immobilized monomeric avidin (Pierce)
  • 2 mM D‐biotin in 10 mM phosphate buffer, pH 7.4 (see appendix 2A)
  • 30% acetonitrile/0.4% formic acid
  • 100 mM glycine‐HCl buffer, pH 2.8,
  • 2× phosphate‐buffered saline (2× PBS; 20 mM phosphate, 300 mM NaCl, pH 7.2; see appendix 2A)
  • 50 mM ammonium bicarbonate /20% methanol
  • Zeba desalting spin columns (Pierce)
  • Microcon YM‐10 ultrafiltration unit (Millipore)
  • Handee Mini‐Spin column (Pierce)
  • Vacuum evaporator (e.g., SpeedVac)
  • Additional reagents and equipment for determining protein concentration ( appendix 3G or )

Support Protocol 1: In‐Gel Trypsin Digestion of Samples

  Materials
  • One‐ or two‐dimensional stained and blotted gels (from parallel electrophoresis; protocol 1 or protocol 2)
  • 50% (v/v) acetonitrile/25 mM ammonium bicarbonate
  • Acetonitrile
  • 0.5 µg/µl trypsin stock solution: 20 µg sequencing‐grade modified porcine trypsin (Promega) in 40 µl of 1% (v/v) acetic acid; store at −80°C
  • 25 mM ammonium bicarbonate (12.5 ng/µl; JT Baker)
  • Scalpel
  • Vacuum evaporator (e.g., SpeedVac)

Support Protocol 2: Desalting Tryptic Peptide Mixtures Using C18 ZipTips

  Materials
  • Dried ARP‐labeled peptide mixture ( protocol 5)
  • 5% (v/v) acetonitrile/0.1% (v/v) trifluoroacetic acid (Fluka)
  • 1% (v/v) trifluoroacetic acid
  • 50% (v/v) acetonitrile/0.1% (w/v) trifluoroacetic acid (Fluka)
  • Acetonitrile
  • C18 ZipTips (Millipore)
  • Matrix solution: 1:1 (v/v) 10 µg/µl α‐cyano‐4‐hydroxy‐cinnamic acid/0.1% trifluoroacetic acid in 50%(v/v) acetonitrile
  • pH paper Vacuum evaporator (e.g., SpeedVac)
  • MALDI target plate (with 144 spots)

Support Protocol 3: Sample Preparation for MALDI MS Analysis Using a Nano‐HPLC Equipped with Target Spotter

  Materials
  • ARP‐labeled protein sample, digested in‐gel ( protocol 5)
  • 1% (v/v) trifluoroacetic acid
  • Acetonitrile
  • α‐cyano‐4‐hydroxycinnamic acid (HCCA; Sigma)
  • 6 mg/ml NH 4H 2PO 4 stock solution
  • 60% (v/v) acetonitrile
  • Autosampler vials
  • 0.2‐µm PTFE syringe filter (Nalgene)
  • FAMOS well‐plate microautosampler
  • Nano‐LC column (3 µm × 150 mm, 100 Å, C18 PepMap100; Dionex)
  • Precolumn (PepMap, 300 µm × 5 mm, 100 Å; Dionex)
  • Probot microfraction collector (Dionex)
  • MALDI target plate

Basic Protocol 4: Mass Spectrometry and Data Analysis

  Materials
  • 4700 calibration mixture (Applied Biosystem)
  • MALDI MS/MS system, e.g., 4700 Proteomic Analyzer (Applied Biosystem)
  • GPS Explorer 3.6 software (Applied Biosystems)
  • MASCOT database searching software (Matrix Science)
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Figures

Videos

Literature Cited

Literature Cited
   Chavez, J., Wu, J., Han, B., Chung, W.G., and Maier, C.S. 2006. New role for an old probe: Affinity labeling of oxylipid protein conjugates by N′‐aminooxymethylcarbonylhydrazino d‐biotin. Anal. Chem. 78:6847‐6854.
   Mirzaei, H. and Regnier, F. 2006. Enrichment of carbonylated peptides using Girard P reagent and strong cation exchange chromatography. Anal. Chem. 78:770‐778.
   Marnett, L.J., Riggins, J.N., and West, J.D. 2003. Endogenous generation of reactive oxidants and electrophiles and their reactions with DNA and protein. J. Clin. Invest. 111:583‐593.
   Requena, J.R., Chao, C.C., Levine, R.L., and Stadtman, E.R. 2001. Glutamic and aminoadipic semialdehydes are the main carbonyl products of metal‐catalyzed oxidation of proteins. Proc. Natl. Acad. Sci. U.S.A. 98:69‐74.
   Sayre, L.M., Smith, M.A., and Perry, G. 2001. Chemistry and biochemistry of oxidative stress in neurodegenerative disease. Curr. Med. Chem. 8:721‐738.
   Spiteller, G. 2001. Lipid peroxidation in aging and age‐dependent diseases. Exp. Gerontol. 36:1425‐1457.
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