A top down approach to protein structural studies using chemical cross-linking and Fourier transform mass spectrometry.
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.
Kruppa
Gary H
GH
Sandia National Laboratories, Livermore, CA 94551-0969, USA. gkruppa@sandia.gov
Schoeniger
Joseph
J
Young
Malin M
MM
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry*; D005583:Fourier Analysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D025801:Ubiquitin / Q000737:chemistry
D003432:Cross-Linking Reagents; D011506:Proteins; D025801:Ubiquitin