This article is a U.S. Government work and is in the public domain in the U.S.A.
A top down approach to protein structural studies using chemical cross-linking and Fourier transform mass spectrometry†
Article first published online: 2 DEC 2002
This article is a US Government work and is in the public domain in the USA. Published in 2002 by John Wiley & Sons, Ltd.
Rapid Communications in Mass Spectrometry
Volume 17, Issue 2, pages 155–162, 30 January 2003
How to Cite
Kruppa, G. H., Schoeniger, J. and Young, M. M. (2003), A top down approach to protein structural studies using chemical cross-linking and Fourier transform mass spectrometry. Rapid Commun. Mass Spectrom., 17: 155–162. doi: 10.1002/rcm.885
- Issue published online: 2 DEC 2002
- Article first published online: 2 DEC 2002
- Manuscript Accepted: 4 NOV 2002
- Manuscript Received: 29 OCT 2002
Mass spectrometric analysis of wild-type proteins that have been covalently modified by bifunctional cross-linking reagents and then digested proteolytically can be used to obtain low-resolution distance constraints, which can be useful for protein structure determination. Limitations of this approach include time-consuming separation steps, such as the separation of internally cross-linked protein monomers from covalent dimers, and a susceptibility to artifacts due to low levels of natural and man-made peptide modifications that can be mistaken for cross-linked species. The results presented here show that when a crude cross-linked protein mixture is injected into an electrospray ionization Fourier transform mass spectrometry (ESI-FTMS) instrument, the cross-link positions can be localized by fragmentation and mass spectrometry on the ‘gas-phase purified’ singly internally cross-linked monomer. Our results show that reaction of ubiquitin with the homobifunctional lysine-lysine cross-linking reagent dissuccinimidyl suberate (DSS) resulted in two cross-links consistent with the known ubiquitin tertiary structure (K6-K11 and K48-K63). Because no protein or peptide chemistry steps are needed, other than the initial cross-linking, this new top down approach appears well suited for high-throughput experiments with multiple cross-linkers and reaction conditions. Published in 2002 by John Wiley & Sons, Ltd.