Targeted Identification of Infection‐Related HLA Class I–Presented Epitopes by Stable Isotope Tagging of Epitopes (SITE)

H.D. Meiring1, E.C. Soethout1, A.P.J.M. de Jong1, C.A.C.M. van Els1

1 Netherlands Vaccine Institute, Bilthoven
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 16.3
DOI:  10.1002/0471142735.im1603s77
Online Posting Date:  May, 2007
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Abstract

Identification of peptides presented in human leukocyte antigen (HLA) class I molecules after viral infection is of strategic importance for immunology and vaccine development. A powerful strategy aimed at the rapid, unambiguous identification of naturally processed HLA class I–associated peptides, which are induced by viral infection, is presented here. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus‐infected cells with stable isotope‐labeled amino acids that are expected to be anchor residues for the human leukocyte antigen allele of interest. Subsequently, these cells are mixed with an equal number of noninfected cells, which are cultured in normal medium. Finally, peptides are acid‐eluted from immunoprecipitated HLA molecules and subjected to two‐dimensional nanoscale liquid chromatography–mass spectrometry analysis. Virus‐induced peptides are identified through computer‐assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules.

Keywords: HLA class I; epitope; mass spectrometry; virus; CD8+ T cell

     
 
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Table of Contents

  • Basic Protocol 1: Epitope Tagging During Culture and Viral Infection of HLA‐Typed Antigen‐Presenting Cells
  • Basic Protocol 2: Isolation of HLA Class I–Presented Peptides from Human Virus‐Infected Cells
  • Basic Protocol 3: Identification of Infection‐Related HLA Class I–Presented Epitopes
  • Support Protocol 1: Epitope Quantification
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Epitope Tagging During Culture and Viral Infection of HLA‐Typed Antigen‐Presenting Cells

  Materials
  • Cells (any cell type that can be infected, HLA class I–typed)
  • Unlabeled amino acids, light
  • Labeled amino acids (98 atom%‐enriched for one or more stable isotopes, 13C, 2H, 15N etc.; Cambridge Isotope Laboratories), heavy
  • Standard growth medium, used for the particular cell population under investigation
  • FBS
  • Virus of known titer
  • CHAPS (3‐[(3‐Cholamidopropyl)dimethylammonio]‐1‐propane‐sulfonate hydrate, Roche)
  • PBS
  • Dry ice/ethanol bath
  • 50‐ml tubes

Basic Protocol 2: Isolation of HLA Class I–Presented Peptides from Human Virus‐Infected Cells

  Materials
  • Cell pellet
  • 1×, 2× lysis buffer with protease inhibitors (see recipe)
  • Sepharose beads (Pharmacia)
  • Sepharose beads coupled to irrelevant antibodies present in normal mouse serum (Pharmacia)
  • 1× lysis buffer with iodocetamide only
  • Sepharose beads coupled to antibodies specific for HLA class I molecules of interest (Pharmacia)
  • Wash buffer A: 20 mM Tris⋅Cl, pH 8.0 and 120 mM NaCl
  • Wash buffer B: 20 mM Tris⋅Cl, pH 8.0 and 1 M NaCl
  • Wash buffer C: 20 mM Tris⋅Cl, pH 8.0
  • Wash buffer D: 10 mM Tris⋅Cl, pH 8.0
  • 10% (v/v) acetic acid
  • Orbital shaker
  • 10‐ml round‐bottom polycarbonate ultracentrifuge tube with sealing caps (Nalgene)
  • Ultracentrifuge, 4°C
  • 0.45‐µm filter
  • Disposable columns (Pierce)
  • 70°C water bath
  • 10‐kD Macrosep filter (Millipore)
  • 50‐ml tubes

Basic Protocol 3: Identification of Infection‐Related HLA Class I–Presented Epitopes

  Materials
  • HLA class I eluate (see protocol 2)
  • Water containing 0.1% (v/v) trifluoroacetic acid (TFA)
  • SCX solvent A (see recipe)
  • Transfer solvent: 1:1 (v/v) water/acetonitrile and 0.5% (v/v) acetic acid
  • SCX solvent B (see recipe)
  • SCX solvent C (see recipe)
  • SCX solvent D (see recipe)
  • Reconstitution (see recipe)
  • HPLC solvent A: water and 0.1 M acetic acid
  • HPLC solvent B: acetonitrile and 0.1 M acetic acid
  • Vacuum centrifuge (Eppendorf)
  • HPLC system comprising of a solvent vacuum degasser, a quaternary pump, an injection system with a 10‐µl sample injection loop and, optionally, a UV‐detector set at 215 nm (see setup in Fig. ); all connecting tubings are 100‐µm i.d. undeactivated fused silica and the columns used are:
    • Trapping column: Hypercarb (7‐µm particle size, 5‐mm length × 200‐µm i.d.; NanoSeparations)
    • Analytical column: PolySULFOETHYL Aspartamide (5‐µm particle size, 300‐Å pore size, 10‐cm length × 200‐µm i.d.; NanoSeparations)
  • 100‐µl microvials or vial inserts that fit the autosampler instrument used in LC‐MS analysis
  • Nanoscale LC system comprising of a solvent vacuum degasser, a high pressure mixing binary pump, an autosampler able to inject 10‐µl sample volume (see setup in Fig. ), all connecting tubings are made of 100‐µm i.d. undeactivated fused silica and the columns used are:
    • Trapping column: Reprosil‐Pur C18‐AQ (5‐µm particle size, 120‐Å pore size, 20‐mm length × 100‐µm i.d.; NanoSeparations)
    • Analytical column: Reprosil‐Pur C18‐AQ (5‐µm particle size, 120‐Å pore size, 25‐cm length × 50‐µm i.d.; NanoSeparations)—column end butt‐connected onto a conductive nanospray tip (25‐µm i.d. fused silica tubing tapered to a 5‐µm i.d. tip, gold/carbon‐coated; NanoSeparations)
  • Tandem mass spectrometer (mass resolution of 10,000 FWHM)
  • MS/MS spectra data interpreting software, e.g., Mascot (Perkins et al., ; http://www.matrixscience.com, Matrix Science), ProteinProspector (http://www.prospector.ucsf.edu, University of California), BioWorks (Thermo Scientific), or ProteinLynx (Waters)

Support Protocol 1: Epitope Quantification

  Materials
  • Synthetic analogs of the identified HLA class I–associated peptides
  • Reconstitution solvent (see recipe)
  • HPLC solvent A: water and 0.1 M acetic acid
  • HPLC solvent B: acetonitrile and 0.1 M acetic acid
  • Nanoscale LC system (see protocol 3)
  • Tandem mass spectrometer (see protocol 3)
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Figures

Videos

Literature Cited

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Key Reference
   Meiring et al., 2006. See above.
  This is the first publication that employs metabolic labeling strategies within the area of HLA class I–associated peptides. It comprehensively presents data on the identification and quantification of infection‐related HLA class I–associated peptides utilizing the SITE strategy with two different pathogens: measles virus and respiratory syncytial virus.
Internet Resources
   http://hlaligand.ouhsc.edu/index_2.html
  HLA Ligand/Motif database.
   http://www.matrixscience.com
  Matrix Science Web site.
   http://www.msmetrix.com
  MS‐Xelerator, accelerating data analysis in LC‐MS profiling studies from MsMetrix.
   http://prospector.ucsf.edu
  Proteomics tools for mining sequence databases in conjunction with mass spectrometry experiments from the University of California, San Francisco.
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