Mapping of protein

protein contact surfaces by hydrogen/deuterium exchange, followed by on-line high-performance liquid chromatography-electrospray ionization fourier-transform ion-cyclotron-resonance mass analysis

TuKiet T. Lam, Jason K. Lanman, Mark Emmett, Christopher L. Hendrickson, Alan G. Marshall, Peter E. Prevelige

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

For protein complexes too large, uncrystallizable/insoluble, or low concentration for conventional X-ray diffraction or nuclear magnetic resonance analysis, the contact surface(s) may be mapped by comparing H/2H exchange rate (and thus solvent accessibility) of backbone amide hydrogens in free vs. complexed protein(s). The protein is first exposed to 2H2O, allowed to exchange for each of several reaction periods, and then digested with pepsin. The extent and rate of H/2H exchange is determined by measuring the increase in mass with H/2H exchange period for each of the peptides. Here, we present an experimental protocol that combines rapid (to minimize back-exchange) HPLC front-end separation with ultrahigh-resolution mass analysis (needed to distinguish the isotopic distributions of dozens of peptides simultaneously). The method is used to study the assembled human immunodeficiency virus type capsid protein (CA) and its soluble form.

Original languageEnglish (US)
Pages (from-to)85-95
Number of pages11
JournalJournal of Chromatography A
Volume982
Issue number1
DOIs
StatePublished - Dec 20 2002
Externally publishedYes

Fingerprint

Cyclotrons
Cyclotron resonance
Electrospray ionization
Deuterium
High performance liquid chromatography
Fourier Analysis
Hydrogen
Fourier transforms
Membrane Proteins
High Pressure Liquid Chromatography
Ions
Human Immunodeficiency Virus Proteins
Peptides
Proteins
Pepsin A
Capsid Proteins
Viruses
Amides
X-Ray Diffraction
Ion exchange

Keywords

  • Capsid
  • Human immunodeficiency virus
  • Hydrogen-deuterium exchange
  • Ion cyclotron resonance
  • Mass spectrometry
  • Proteins
  • Viruses

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Mapping of protein : protein contact surfaces by hydrogen/deuterium exchange, followed by on-line high-performance liquid chromatography-electrospray ionization fourier-transform ion-cyclotron-resonance mass analysis. / Lam, TuKiet T.; Lanman, Jason K.; Emmett, Mark; Hendrickson, Christopher L.; Marshall, Alan G.; Prevelige, Peter E.

In: Journal of Chromatography A, Vol. 982, No. 1, 20.12.2002, p. 85-95.

Research output: Contribution to journalArticle

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AU - Hendrickson, Christopher L.

AU - Marshall, Alan G.

AU - Prevelige, Peter E.

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