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Proteins are dynamic entities constantly moving and altering their structures based on their functions and interactions inside and outside the cell. Formaldehyde cross-linking combined with mass spectrometry can accurately capture interactions of these rapidly changing biomolecules while maintaining their physiological surroundings. Even with its numerous established uses in biology and compatibility...
With the continuing trend to study larger and more complex systems, the application of protein cross-linking coupled with mass spectrometry (XL-MS) provides a varied toolkit perfectly suited to achieve these goals. By freezing the transient interactions through the formation of covalent bonds, XL-MS provides a vital insight into both the structure and organization of proteins in a wide variety of...
Recent development of high-resolution mass spectrometry (MS) instruments enables chemical crosslinking (XL) to become a high-throughput method for obtaining structural information about proteins. Restraints derived from XL-MS experiments have been used successfully for structure refinement and protein–protein docking. However, one formidable question is under which circumstances XL-MS data might be...
Top-down sequencing methods are becoming increasingly relevant for protein characterization, in particular electron capture (ECD) and electron transfer dissociation (ETD) which allow for extensive backbone cleavage with minimal side reactions. The ability to obtain sequence-specific fragments while maintaining aspects of the higher-order structure, as well as the position of deuterium labels in H/D...
Chemical cross-linking is a promising technology for protein tertiary structure determination. Though the data has low spatial resolution, it is possible to obtain it at physiological conditions on proteins that are not amenable to standard high resolution techniques such as X-ray, NMR analysis and cryo-EM. Here we demonstrate the utilization of isotopically labeled chemical cross-linking to visualize...
We developed a limited proteolysis assay for estimating dynamics in plasma-borne protease activities using MALDI ToF MS analysis as readout. A highly specific limited proteolysis activity was elicited in human plasma by shifting the pH to 6. Mass spectrometry showed that two singly charged ion signals at m/z 2753.44 and m/z 2937.56 significantly increased in abundance under mild acidic conditions...
Protein–protein interactions play a central role in the regulation of many biochemical processes (e.g. the system participating in enzyme catalysis). Therefore, a deeper understanding of protein–protein interactions may contribute to the elucidation of many biologically important mechanisms. For this purpose, it is necessary to establish the composition and stoichiometry of supramolecular complexes...
Photo-induced cross-linking is a highly promising technique to investigate protein conformations and protein–protein interactions in their natural cellular environment. One strategy relies on the non-directed incorporation of diazirine-containing photo-activatable amino acids into proteins and a subsequent cross-link formation induced by UV-A irradiation. The advantage of this photo-cross-linking...
Ribonucleoprotein (RNP) complexes play important roles in the cell by mediating basic cellular processes, including gene expression and its regulation. Understanding the molecular details of these processes requires the identification and characterization of protein–RNA interactions. Over the years various approaches have been used to investigate these interactions, including computational analyses...
The recruitment of different chemokines and growth factors by glycosaminoglycans (GAGs) such as chondroitin sulfate or hyaluronan plays a critical role in wound healing processes. Thus, there is a special interest in the design of artificial extracellular matrices with improved properties concerning GAG interaction with common regulating proteins. In this study, amide hydrogen/deuterium (H/D) exchange...
Structural mass spectrometry (MS) is a field with growing applicability for addressing complex biophysical questions regarding proteins and protein complexes. One of the major structural MS approaches involves the use of chemical cross-linking coupled with MS analysis (CX-MS) to identify proximal sites within macromolecules. Identified cross-linked sites can be used to probe novel protein–protein...
Disulfide bonds are valuable constraints in protein structure modeling. The Cys–Cys disulfide bond undergoes specific fragmentation under CID and, therefore, can be considered as a CID-cleavable crosslink. We have recently reported on the benefits of using non-specific digestion with proteinase K for inter-peptide crosslink determination. Here, we describe an updated application of our CID-cleavable...
With the development of soft ionization techniques such as electrospray ionization (ESI), mass spectrometry (MS) has found widespread application in structural biology. The ability to transfer large biomolecular complexes intact into the gas-phase, combined with the low sample consumption and high sensitivity of MS, has made ESI-MS a method of choice for the characterization of macromolecules. This...
Structural mass spectrometry encompasses an increasing range of methods aimed at collecting as much structural information as possible on a biomolecule or its related complexes. Originally limited to the analysis of the primary structures of proteins, mass spectrometry has evolved over the past 20years to provide information on the secondary, tertiary and even quaternary structure of proteins. Furthermore,...
In this article, we describe our methods and protocols using collision-induced dissociative chemical crosslinking-tandem mass spectrometry (CID-CXL-MS/MS) analysis and the practical considerations when implementing these reagents and methodology for protein crosslinking studies. The synthesis of our novel chemical crosslinkers is described as well as their use for effectively labeling protein and...
Characterisation of the conformational states adopted during protein folding, including globally unfolded/disordered structures and partially folded intermediate species, is vital to gain fundamental insights into how a protein folds. In this work we employ fast photochemical oxidation of proteins (FPOP) to map the structural changes that occur in the folding of the four-helical bacterial immunity...
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