“Out-Gel” Tryptic Digestion Procedure for Chemical Cross-Linking Studies with Mass Spectrometric Detection

2014 ◽  
Vol 13 (2) ◽  
pp. 527-535 ◽  
Author(s):  
Evgeniy V. Petrotchenko ◽  
Jason J. Serpa ◽  
Ashley N. Cabecinha ◽  
Mary Lesperance ◽  
Christoph H. Borchers
Keyword(s):  
Mass Spectrometric ◽  
Mass Spectrometric Detection ◽  
Tryptic Digestion ◽  
Cross Linking ◽  
Digestion Procedure ◽  
Chemical Cross Linking

scholarly journals Chemical cross-linking analyses of ox neurofilaments

1986 ◽  
Vol 234 (3) ◽  
pp. 587-591 ◽  
Author(s):  
M J Carden ◽  
P A M Eagles
Keyword(s):  
Ionic Strength ◽  
Tryptic Digestion ◽  
Cross Linking ◽  
Cross Link ◽  
Close Proximity ◽  
Neurofilament Polypeptides ◽  
Phenanthroline Complex ◽  
Low Ionic Strength ◽  
Polypeptide Chains ◽  
Chemical Cross Linking

Freshly isolated intact ox neurofilaments have been incubated with copper(II)-o-phenanthroline complex to induce thiol cross-linking between the two largest (apparent Mr 205 000 and 158 000) polypeptide components. Subsequent tryptic digestion shows that the thiol bonds formed between these polypeptides are distributed exclusively among ‘rod-domain’ fragments that remain associated with intact sedimentable filaments. These observations suggest that the polypeptide chains of the two largest neurofilament components are closely arranged within the backbone but are separate from one another in more peripheral regions. Soluble protofilaments derived from neurofilament disassembly at low ionic strength and high pH have also been cross-linked via thiol bonds in order to determine the polypeptide arrangement within these structures. All three neurofilament polypeptides cross-link more readily when in the form of protofilaments than when in the form of fully assembled filaments, and the pattern of cross-linked complexes formed is different. Analysis of one of these complexes shows that at least some of the protofilaments are composed of oligomers containing both the 72 000- and the 158 000-Mr neurofilament polypeptides arranged in close proximity.

scholarly journals Chemical cross-linking with thiol-cleavable reagents combined with differential mass spectrometric peptide mapping-A novel approach to assess intermolecular protein contacts

2000 ◽  
Vol 9 (8) ◽  
pp. 1503-1518 ◽  
Author(s):  
Keiryn L. Bennett ◽  
Martin Kussmann ◽  
Marie Mikkelsen ◽  
Peter Roepstorff ◽  
Per Björk ◽  
...  
Keyword(s):  
Peptide Mapping ◽  
Mass Spectrometric ◽  
Cross Linking ◽  
Novel Approach ◽  
Chemical Cross Linking

Statistical Force-Field for Structural Modeling Using Chemical Cross-Linking/mass Spectrometry Distance Constraints

2018 ◽  
Author(s):  
Allan J. R. Ferrari ◽  
Fabio C. Gozzo ◽  
Leandro Martinez
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Force Field ◽  
Protein Structures ◽  
Protein Structure Determination ◽  
Structural Modeling ◽  
Cross Linking ◽  
Amino Acid Residues ◽  
Distance Constraints ◽  
Chemical Cross Linking

<div><p>Chemical cross-linking/Mass Spectrometry (XLMS) is an experimental method to obtain distance constraints between amino acid residues, which can be applied to structural modeling of tertiary and quaternary biomolecular structures. These constraints provide, in principle, only upper limits to the distance between amino acid residues along the surface of the biomolecule. In practice, attempts to use of XLMS constraints for tertiary protein structure determination have not been widely successful. This indicates the need of specifically designed strategies for the representation of these constraints within modeling algorithms. Here, a force-field designed to represent XLMS-derived constraints is proposed. The potential energy functions are obtained by computing, in the database of known protein structures, the probability of satisfaction of a topological cross-linking distance as a function of the Euclidean distance between amino acid residues. The force-field can be easily incorporated into current modeling methods and software. In this work, the force-field was implemented within the Rosetta ab initio relax protocol. We show a significant improvement in the quality of the models obtained relative to current strategies for constraint representation. This force-field contributes to the long-desired goal of obtaining the tertiary structures of proteins using XLMS data. Force-field parameters and usage instructions are freely available at http://m3g.iqm.unicamp.br/topolink/xlff <br></p></div><p></p><p></p>

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