Abstract
The fundamentals of how protein–protein/RNA/DNA interactions influence the structures and functions of the workhorses from the cells have been well documented in the 20th century. A diverse set of methods exist to determine such interactions between different components, particularly, the mass spectrometry (MS) methods, with its advanced instrumentation, has become a significant approach to analyze a diverse range of biomolecules, as well as bring insights to their biomolecular processes. This review highlights the principal role of chemistry in MS-based structural proteomics approaches, with a particular focus on the chemical cross-linking of protein–protein/DNA/RNA complexes. In addition, we discuss different methods to prepare the cross-linked samples for MS analysis and tools to identify cross-linked peptides. Cross-linking mass spectrometry (CLMS) holds promise to identify interaction sites in larger and more complex biological systems. The typical CLMS workflow allows for the measurement of the proximity in three-dimensional space of amino acids, identifying proteins in direct contact with DNA or RNA, and it provides information on the folds of proteins as well as their topology in the complexes. Principal CLMS applications, its notable successes, as well as common pipelines that bridge proteomics, molecular biology, structural systems biology, and interactomics are outlined.
Highlights
Decades of research into the cell biology, molecular biology, biochemistry, structural biology, and biophysics have produced a detailed understanding of individualDNA/RNA/protein molecules, and their interconnected networks
In the cross-linking mass spectrometry (CLMS) approach, chemical cross-linking reagents are used to join the components of interacting complexes, followed by LC-MS/MS (Liquid chromatography-tandem mass spectrometry) analysis that enables in vivo and in vitro methods to define the native PPIs of a protein complex, under optimal conditions
Growing technologies to map out interactions between protein–protein
Summary
Decades of research into the cell biology, molecular biology, biochemistry, structural biology, and biophysics have produced a detailed understanding of individual. Quantitative cross-linking mass spectrometry (QCLMS) approaches investigate protein structures as well as the dynamics of their interactions [46,47,48,49,50,51]. Yu et al [56], implemented the TMT approach in a multiplexed comparison of protein complex dynamics and protein–protein interactions Their QMIX (Quantitation of Multiplexed, Isobaric-labeled cross (X)-linked peptides) workflow with TMT labeling, achieves peptide quantitation from MS3 data that eliminates interference from ions that were observed in MS1 data along with isotope labeled cross-linkers or SILAC cross-linking [56]. A limitation of using chemical cross-linking while using these methods is the high risk of detecting non-specific interactions These limitations can be addressed using more than one cross-linker of differing activities or spanning various distances and by varying the ratio of reagent to protein complex. The various workflows that have been developed to implement CLMS represent a vast toolkit that can help to provide novel insight into the structure and organization of proteins in order to define protein–protein interactions and probing PPI interfaces
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