RESUMO
We present a concise workflow to enhance the mass spectrometric detection of crosslinked peptides by introducing sequential digestion and the crosslink identification software xiSEARCH. Sequential digestion enhances peptide detection by selective shortening of long tryptic peptides. We demonstrate our simple 12-fraction protocol for crosslinked multi-protein complexes and cell lysates, quantitative analysis, and high-density crosslinking, without requiring specific crosslinker features. This overall approach reveals dynamic protein-protein interaction sites, which are accessible, have fundamental functional relevance and are therefore ideally suited for the development of small molecule inhibitors.
Assuntos
Espectrometria de Massas/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas/química , Proteômica/métodos , Citosol/química , Humanos , Células K562 , Modelos Moleculares , Fragmentos de Peptídeos/química , Conformação Proteica , SoftwareRESUMO
SUMMARY: Proteins frequently function as parts of complexes, assemblages of multiple proteins and other biomolecules, yet network visualizations usually only show proteins as parts of binary interactions. ComplexViewer visualizes interactions with more than two participants and thereby avoids the need to first expand these into multiple binary interactions. Furthermore, if binding regions between molecules are known then these can be displayed in the context of the larger complex. AVAILABILITY AND IMPLEMENTATION: freely available under Apache version 2 license; EMBL-EBI Complex Portal: http://www.ebi.ac.uk/complexportal; Source code: https://github.com/MICommunity/ComplexViewer; Package: https://www.npmjs.com/package/complexviewer; http://biojs.io/d/complexviewer. Language: JavaScript; Web technology: Scalable Vector Graphics; Libraries: D3.js. CONTACT: colin.combe@ed.ac.uk or juri.rappsilber@ed.ac.uk.
Assuntos
Biologia Computacional/métodos , Modelos Biológicos , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Software , Substâncias Macromoleculares/metabolismo , Ligação ProteicaRESUMO
UNLABELLED: xiNET is a visualization tool for exploring cross-linking/mass spectrometry results. The interactive maps of the cross-link network that it generates are a type of node-link diagram. In these maps xiNET displays: (1) residue resolution positional information including linkage sites and linked peptides; (2) all types of cross-linking reaction product; (3) ambiguous results; and, (4) additional sequence information such as domains. xiNET runs in a browser and exports vector graphics which can be edited in common drawing packages to create publication quality figures. AVAILABILITY: xiNET is open source, released under the Apache version 2 license. Results can be viewed by uploading data to http://crosslinkviewer.org/ or by downloading the software from http://github.com/colin-combe/crosslink-viewer and running it locally.
Assuntos
Aminoácidos/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , Mapas de Interação de Proteínas , Software , Espectrometria de Massas , Multimerização Proteica , Estrutura Terciária de Proteína , Fatores de Transcrição TFII/química , Fatores de Transcrição TFII/metabolismoRESUMO
Cross-linking mass spectrometry (MS) has substantially matured as a method over the past 2 decades through parallel development in multiple labs, demonstrating its applicability to protein structure determination, conformation analysis, and mapping protein interactions in complex mixtures. Cross-linking MS has become a much-appreciated and routinely applied tool, especially in structural biology. Therefore, it is timely that the community commits to the development of methodological and reporting standards. This white paper builds on an open process comprising a number of events at community conferences since 2015 and identifies aspects of Cross-linking MS for which guidelines should be developed as part of a Cross-linking MS standards initiative.