Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein–protein interactions

2006 ◽  
Vol 25 (4) ◽  
pp. 663-682 ◽  
Author(s):  
Andrea Sinz
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Protein Structures ◽  
Three Dimensional ◽  
Cross Linking ◽  
Protein Protein Interactions ◽  
Chemical Cross Linking

scholarly journals Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Cross-Linking Mass Spectrometry

2021 ◽  
Author(s):  
Dmitri R. Davydov ◽  
Bikash Dangi ◽  
Guihua Yue ◽  
Bhagwat Prasad ◽  
Viktor G. Zgoda
Keyword(s):  
Mass Spectrometry ◽  
Cytochrome P450 ◽  
Protein Interactions ◽  
Human Liver ◽  
Liver Microsomes ◽  
Human Liver Microsomes ◽  
Cross Linking ◽  
Cytochrome P450 2E1 ◽  
Protein Protein Interactions ◽  
Chemical Cross Linking

This study aimed on exploration of the system-wide effects of the alcohol-induced increase in the content of cytochrome P450 2E1 (CYP2E1) in the human liver on drug metabolism. Using membrane incorporation of purified CYP2E1 modified with photoreactive crosslinkers benzophenone-4-maleimide (BPM) and 4-(N-succinimidylcarboxy)benzophenone (BPS), we explored the array of its protein-protein interactions (proteome) in human liver microsomes (HLM) with chemical cross-linking mass spectrometry (CXMS). Exposure of bait-incorporated HLM samples to light was followed by isolation of the His-tagged bait protein and its cross-linked aggregates on Ni-NTA agarose. Analyzing the individual bands of SDS-PAGE slabs of thereby isolated protein with the toolset of untargeted proteomics, we detected the cross-linked dimeric and trimeric complexes of CYP2E1 with other drug-metabolizing enzymes. Among the most extensively cross-linked partners of CYP2E1 are cytochromes P450 2A6, 3A4, 2C9, and 4A11. We also detected the conjugates of CYP2E1 with UDP-glucuronosyltransferases (UGTs) 1A6, 1A9, 2B4, 2B15, and 2B17. These results demonstrate the exploratory power of the proposed CXMS strategy and corroborate the concept of tight functional integration in the human drug-metabolizing ensemble through protein-protein interactions of the constituting enzymes. Of particular interest is the observation of efficient cross-linking of CYP2E1 with CYP4A11. This enzyme plays a central role in the synthesis of vasoactive eicosanoids and its interactions with alcohol-inducible CYP2E1 may shed light on the mechanisms of alcohol-induced hypertension.

scholarly journals Identification of Protein-Protein Interactions and Topologies in Living Cells with Chemical Cross-linking and Mass Spectrometry

2008 ◽  
Vol 8 (3) ◽  
pp. 409-420 ◽  
Author(s):  
Haizhen Zhang ◽  
Xiaoting Tang ◽  
Gerhard R. Munske ◽  
Nikola Tolic ◽  
Gordon A. Anderson ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Living Cells ◽  
Cross Linking ◽  
Protein Protein Interactions ◽  
Chemical Cross Linking

scholarly journals Sequential digestion with Trypsin and Elastase in cross-linking/mass spectrometry

2018 ◽  
Author(s):  
Therese Dau ◽  
Kapil Gupta ◽  
Imre Berger ◽  
Juri Rappsilber
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Protein Structures ◽  
Cross Linking ◽  
Cleavage Sites ◽  
Protein Modelling ◽  
Protein Protein Interactions ◽  
Important Approach ◽  
Cross Links ◽  
Sequential Digestion

ABSTRACTCross-linking/mass spectrometry has become an important approach for studying protein structures and protein-protein interactions. The amino acid composition of some protein regions impedes the detection of cross-linked residues, although it would yield invaluable information for protein modelling. Here, we report on a sequential digestion strategy with trypsin and elastase to penetrate regions with a low density of trypsin cleavage sites. We exploited intrinsic substrate recognition properties of elastase to specifically target larger tryptic peptides. Our application of this protocol to the TAF4-12 complex allowed us to identify cross-links in previously inaccessible regions.

scholarly journals Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry

2017 ◽  
Vol 114 (7) ◽  
pp. 1732-1737 ◽  
Author(s):  
Devin K. Schweppe ◽  
Juan D. Chavez ◽  
Chi Fung Lee ◽  
Arianne Caudal ◽  
Shane E. Kruse ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Mitochondrial Function ◽  
Mitochondrial Protein ◽  
Protein Structures ◽  
Protein Docking ◽  
Molecular Probes ◽  
Cross Linking ◽  
Chemical Cross Linking

Mitochondrial protein interactions and complexes facilitate mitochondrial function. These complexes range from simple dimers to the respirasome supercomplex consisting of oxidative phosphorylation complexes I, III, and IV. To improve understanding of mitochondrial function, we used chemical cross-linking mass spectrometry to identify 2,427 cross-linked peptide pairs from 327 mitochondrial proteins in whole, respiring murine mitochondria. In situ interactions were observed in proteins throughout the electron transport chain membrane complexes, ATP synthase, and the mitochondrial contact site and cristae organizing system (MICOS) complex. Cross-linked sites showed excellent agreement with empirical protein structures and delivered complementary constraints for in silico protein docking. These data established direct physical evidence of the assembly of the complex I–III respirasome and enabled prediction of in situ interfacial regions of the complexes. Finally, we established a database and tools to harness the cross-linked interactions we observed as molecular probes, allowing quantification of conformation-dependent protein interfaces and dynamic protein complex assembly.

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