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Title: Native Electrospray and Electron-Capture Dissociation FTICR Mass Spectrometry for Top-Down Studies of Protein Assemblies

Abstract

The high sensitivity, extended mass range, and fast data acquisition/processing of mass spectrometry and its coupling with native electrospray ionization (ESI) make the combination complementary to other biophysical methods of protein analysis. Protein assemblies with molecular masses up to MDa are now accessible by this approach. Most current approaches have used quadrupole/time-of-flight tandem mass spectrometry, sometimes coupled with ion mobility, to reveal stoichiometry, shape, and dissociation of protein assemblies. The amino-acid sequence of the subunits, however, still relies heavily on independent bottom-up proteomics. We describe here an approach to study protein assemblies that integrates electron-capture dissociation (ECD), native ESI, and FTICR mass spectrometry (12 T). Flexible regions of assembly subunits of yeast alcohol dehydrogenase (147 kDa), concanavalin A (103 kDa), and photosynthetic Fenna–Matthews–Olson antenna protein complex (140 kDa) can be sequenced by ECD or “activated-ion” ECD. Furthermore, noncovalent metal-binding sites can also be determined for the concanavalin A assembly. Most importantly, the regions that undergo fragmentation, either from one of the termini by ECD or from the middle of a protein, as initiated by CID, correlate well with the B-factor from X-ray crystallography of that protein. This factor is a measure of the extent an atom can move from itsmore » coordinated position as a function of temperature or crystal imperfections. The approach provides not only top-down proteomics information of the complex subunits but also structural insights complementary to those obtained by ion mobility.« less

Authors:
 [1];  [1];  [2];  [2];  [1]
  1. Washington Univ., St. Louis, MO (United States). Dept. of Chemistry
  2. Washington Univ., St. Louis, MO (United States). Dept. of Chemistry and Dept. of Biology
Publication Date:
Research Org.:
Energy Frontier Research Centers (EFRC); Photosynthetic Antenna Research Center (PARC)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1065573
DOE Contract Number:  
SC0001035
Resource Type:
Journal Article
Journal Name:
Analytical Chemistry
Additional Journal Information:
Journal Volume: 83; Journal Issue: 14; Related Information: PARC partners with Washington University in St. Louis (lead); University of California, Riverside; University of Glasgow, UK; Los Alamos National Laboratory; University of New Mexico; New Mexico Corsortium; North Carolina State University; Northwestern University; Oak Ridge National Laboratory; University of Pennsylvania; Sandia National Laboratories; University of Sheffield, UK; Journal ID: ISSN 0003-2700
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; solar (fuels), photosynthesis (natural and artificial), biofuels (including algae and biomass), bio-inspired, charge transport, membrane, synthesis (novel materials), synthesis (self-assembly)

Citation Formats

Zhang, Hao, Cui, Weidong, Wen, Jianzhong, Blankenship, Robert E., and Gross, Michael L. Native Electrospray and Electron-Capture Dissociation FTICR Mass Spectrometry for Top-Down Studies of Protein Assemblies. United States: N. p., 2011. Web. doi:10.1021/ac200695d.
Zhang, Hao, Cui, Weidong, Wen, Jianzhong, Blankenship, Robert E., & Gross, Michael L. Native Electrospray and Electron-Capture Dissociation FTICR Mass Spectrometry for Top-Down Studies of Protein Assemblies. United States. doi:10.1021/ac200695d.
Zhang, Hao, Cui, Weidong, Wen, Jianzhong, Blankenship, Robert E., and Gross, Michael L. Fri . "Native Electrospray and Electron-Capture Dissociation FTICR Mass Spectrometry for Top-Down Studies of Protein Assemblies". United States. doi:10.1021/ac200695d.
@article{osti_1065573,
title = {Native Electrospray and Electron-Capture Dissociation FTICR Mass Spectrometry for Top-Down Studies of Protein Assemblies},
author = {Zhang, Hao and Cui, Weidong and Wen, Jianzhong and Blankenship, Robert E. and Gross, Michael L.},
abstractNote = {The high sensitivity, extended mass range, and fast data acquisition/processing of mass spectrometry and its coupling with native electrospray ionization (ESI) make the combination complementary to other biophysical methods of protein analysis. Protein assemblies with molecular masses up to MDa are now accessible by this approach. Most current approaches have used quadrupole/time-of-flight tandem mass spectrometry, sometimes coupled with ion mobility, to reveal stoichiometry, shape, and dissociation of protein assemblies. The amino-acid sequence of the subunits, however, still relies heavily on independent bottom-up proteomics. We describe here an approach to study protein assemblies that integrates electron-capture dissociation (ECD), native ESI, and FTICR mass spectrometry (12 T). Flexible regions of assembly subunits of yeast alcohol dehydrogenase (147 kDa), concanavalin A (103 kDa), and photosynthetic Fenna–Matthews–Olson antenna protein complex (140 kDa) can be sequenced by ECD or “activated-ion” ECD. Furthermore, noncovalent metal-binding sites can also be determined for the concanavalin A assembly. Most importantly, the regions that undergo fragmentation, either from one of the termini by ECD or from the middle of a protein, as initiated by CID, correlate well with the B-factor from X-ray crystallography of that protein. This factor is a measure of the extent an atom can move from its coordinated position as a function of temperature or crystal imperfections. The approach provides not only top-down proteomics information of the complex subunits but also structural insights complementary to those obtained by ion mobility.},
doi = {10.1021/ac200695d},
journal = {Analytical Chemistry},
issn = {0003-2700},
number = 14,
volume = 83,
place = {United States},
year = {2011},
month = {7}
}