Premium
A top down approach to protein structural studies using chemical cross‐linking and Fourier transform mass spectrometry
Author(s) -
Kruppa Gary H.,
Schoeniger Joseph,
Young Malin M.
Publication year - 2002
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.885
Subject(s) - chemistry , mass spectrometry , fourier transform ion cyclotron resonance , electrospray ionization , reagent , monomer , covalent bond , fragmentation (computing) , lysine , top down proteomics , chromatography , bifunctional , protein mass spectrometry , organic chemistry , amino acid , biochemistry , polymer , computer science , operating system , catalysis
Abstract 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.