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

Tu Kiet T. Lam; Jason K. Lanman; Mark R. Emmett; Christopher L. Hendrickson; Alan G. Marshall; Peter E. Prevelige

doi:10.1016/S0021-9673(02)01357-2

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

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

Research output: Contribution to journal › Article › peer-review

47 Scopus citations

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 language	English (US)
Pages (from-to)	85-95
Number of pages	11
Journal	Journal of Chromatography A
Volume	982
Issue number	1
DOIs	https://doi.org/10.1016/S0021-9673(02)01357-2
State	Published - Dec 20 2002
Externally published	Yes

Keywords

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

ASJC Scopus subject areas

Analytical Chemistry
Biochemistry
Organic Chemistry

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10.1016/S0021-9673(02)01357-2

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Lam, T. K. T., Lanman, J. K., Emmett, M. R., Hendrickson, C. L., Marshall, A. G., & Prevelige, P. E. (2002). 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. Journal of Chromatography A, 982(1), 85-95. https://doi.org/10.1016/S0021-9673(02)01357-2

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, Tu Kiet T.; Lanman, Jason K.; Emmett, Mark R. et al.
In: Journal of Chromatography A, Vol. 982, No. 1, 20.12.2002, p. 85-95.

Research output: Contribution to journal › Article › peer-review

Lam, TKT, Lanman, JK, Emmett, MR, Hendrickson, CL, Marshall, AG & Prevelige, PE 2002, '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', Journal of Chromatography A, vol. 982, no. 1, pp. 85-95. https://doi.org/10.1016/S0021-9673(02)01357-2

Lam TKT, Lanman JK, Emmett MR, Hendrickson CL, Marshall AG, Prevelige PE. 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. Journal of Chromatography A. 2002 Dec 20;982(1):85-95. doi: 10.1016/S0021-9673(02)01357-2

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title = "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",

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.",

keywords = "Capsid, Human immunodeficiency virus, Hydrogen-deuterium exchange, Ion cyclotron resonance, Mass spectrometry, Proteins, Viruses",

author = "Lam, {Tu Kiet T.} and Lanman, {Jason K.} and Emmett, {Mark R.} and Hendrickson, {Christopher L.} and Marshall, {Alan G.} and Prevelige, {Peter E.}",

note = "Funding Information: We acknowledge John Quinn{\textquoteright}s technical assistance and give special thanks to David Horn and Greg Blakney for providing thrash and modification to midas analysis to facilitate monoisotopic peak determination and centroid determination. Work supported by NSF (CHE-99-09502), NIH (GM-31683; AI-44626), Florida State University, and the National High Magnetic Field Laboratory in Tallahassee, FL.",

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AU - Emmett, Mark R.

AU - Hendrickson, Christopher L.

AU - Marshall, Alan G.

AU - Prevelige, Peter E.

N1 - Funding Information: We acknowledge John Quinn’s technical assistance and give special thanks to David Horn and Greg Blakney for providing thrash and modification to midas analysis to facilitate monoisotopic peak determination and centroid determination. Work supported by NSF (CHE-99-09502), NIH (GM-31683; AI-44626), Florida State University, and the National High Magnetic Field Laboratory in Tallahassee, FL.

PY - 2002/12/20

Y1 - 2002/12/20

N2 - 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.

AB - 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.

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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

Abstract

Keywords

ASJC Scopus subject areas

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