Lysine-specific chemical cross-linking of protein complexes and identification of cross-linking sites using LC-MS/MS and the xQuest/xProphet software pipeline

2013 ◽  
Vol 9 (1) ◽  
pp. 120-137 ◽  
Author(s):  
Alexander Leitner ◽  
Thomas Walzthoeni ◽  
Ruedi Aebersold
Keyword(s):  
Protein Complexes ◽  
Cross Linking ◽  
Software Pipeline ◽  
Specific Chemical ◽  
Chemical Cross Linking

scholarly journals Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes

2014 ◽  
Vol 111 (26) ◽  
pp. 9455-9460 ◽  
Author(s):  
Alexander Leitner ◽  
Lukasz A. Joachimiak ◽  
Pia Unverdorben ◽  
Thomas Walzthoeni ◽  
Judith Frydman ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Complexes ◽  
Cross Linking ◽  
Acidic Residues ◽  
Chemical Cross Linking

scholarly journals Influence of Cross-Linker Polarity on Selectivity towards Lysine Side Chains

2020 ◽  
Author(s):  
Jan Fiala ◽  
Zdeněk Kukačka ◽  
Petr Novák
Keyword(s):  
Structural Information ◽  
Protein Complexes ◽  
Water Soluble ◽  
Cross Linking ◽  
Side Chains ◽  
Model Protein ◽  
Cross Linker ◽  
Progressive Technology ◽  
The Impact ◽  
Chemical Cross Linking

The combination of chemical cross-linking and mass spectrometry is currently a progressive technology for deriving structural information of proteins and protein complexes. In addition, chemical cross-linking is a powerful tool for stabilizing macromolecular complexes for single particle cryo-electron microscopy. Broad pallets of cross-linking chemistry, currently available for the majority of cross-linking experiments, still rely on the amine-reactive N-hydroxysuccinimide esters targeting mainly N-termini and lysine side chains. These cross-linkers are divided into two groups: water soluble and water insoluble; and research teams prefer one or another speculating on the benefits of their choice. However, the effect of cross-linker polarity on the outcome of cross-linking reaction has never been studied. Herein, we use both polar (bis(sulfosuccinimidyl) glutarate) and non-polar (disuccinimidyl glutarate) cross-linkers and systematically investigated the impact of cross-linker hydrophobicity on resulting distance constraints, using bovine serum albumin as a model protein.

scholarly journals Proper evaluation of chemical cross-linking-based spatial restraints improves the precision of modeling homo-oligomeric protein complexes

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Aljaž Gaber ◽  
Gregor Gunčar ◽  
Miha Pavšič
Keyword(s):  
Quaternary Structure ◽  
Comprehensive Evaluation ◽  
Protein Complexes ◽  
Scoring Function ◽  
Cross Linking ◽  
High Resolution Data ◽  
Oligomeric Proteins ◽  
Oligomeric Protein ◽  
Accessible Surface ◽  
Chemical Cross Linking

Abstract Background The function of oligomeric proteins is inherently linked to their quaternary structure. In the absence of high-resolution data, low-resolution information in the form of spatial restraints can significantly contribute to the precision and accuracy of structural models obtained using computational approaches. To obtain such restraints, chemical cross-linking coupled with mass spectrometry (XL-MS) is commonly used. However, the use of XL-MS in the modeling of protein complexes comprised of identical subunits (homo-oligomers) is often hindered by the inherent ambiguity of intra- and inter-subunit connection assignment. Results We present a comprehensive evaluation of (1) different methods for inter-residue distance calculations, and (2) different approaches for the scoring of spatial restraints. Our results show that using Solvent Accessible Surface distances (SASDs) instead of Euclidean distances (EUCs) greatly reduces the assignation ambiguity and delivers better modeling precision. Furthermore, ambiguous connections should be considered as inter-subunit only when the intra-subunit alternative exceeds the distance threshold. Modeling performance can also be improved if symmetry, characteristic for most homo-oligomers, is explicitly defined in the scoring function. Conclusions Our findings provide guidelines for proper evaluation of chemical cross-linking-based spatial restraints in modeling homo-oligomeric protein complexes, which could facilitate structural characterization of this important group of proteins.

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