Abstract
The unique and remarkable physicochemical properties of protein surface topologies give rise to highly specific biomolecular interactions, which form the framework through which living systems are able to carry out their vast array of functions. Technological limitations undermine efforts to probe protein structures and interactions within unperturbed living systems on a large scale. Rapid chemical stabilization of proteins and protein complexes through chemical cross-linking offers the alluring possibility to study details of the protein structure to function relationships as they exist within living cells. Here we apply the latest technological advances in chemical cross-linking combined with mass spectrometry to study protein topologies and interactions from living human cells identifying a total of 368 cross-links. These include cross-links from all major cellular compartments including membrane, cytosolic and nuclear proteins. Intraprotein and interprotein cross-links were also observed for core histone proteins, including several cross-links containing post-translational modifications which are known histone marks conferring distinct epigenetic functions. Excitingly, these results demonstrate the applicability of cross-linking to make direct topological measurements on post-translationally modified proteins. The results presented here provide new details on the structures of known multi-protein complexes as well as evidence for new protein-protein interactions.
Highlights
Chemical cross-linking has long been used as a method of fixation to preserve biological samples in the fields of histology and pathology [1]
Chemical cross-linking with mass spectrometry (XL-MS)1 is emerging as a powerful technology to study protein structures and interactions in complex biological systems [5]
The use of XL-MS to study protein structures and interactions directly from living cells was first demonstrated on the bacterial system Shewanella oneidensis, where new details on the membrane protein electron transport machinery used during anaerobic respiration were discovered [14]
Summary
Chemical cross-linking has long been used as a method of fixation to preserve biological samples in the fields of histology and pathology [1]. Chemical cross-linking with mass spectrometry (XL-MS) is emerging as a powerful technology to study protein structures and interactions in complex biological systems [5]. Technological advances in chemistry, analytical instrumentation, and informatics are beginning to allow the successful application of XL-MS to study protein topologies and interactions on a large scale in complex biological systems These methods are able to provide low resolution spatial information on protein topologies through distance constraints imposed by the chemical linker arm distance. One application of XL-MS that has seen recent success is the isolation of protein complexes from cells followed by in vitro cross-linking to study the protein complex architecture as demonstrated on the E. coli ribosome and human phosphatase 2A [11, 12] These applications illustrate the powerful utility XL-MS methods hold for acquiring topological data on complexes that are not completely amenable to interrogation via conventional structural biology techniques. Of the 115 identified cross-linked sites obtained from histone proteins 56 were found to contain one or more
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