Protein Interactions, Post-translational Modifications and Topologies in Human Cells

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. Proteins are the principal operatives within cells, involved in carrying out essentially all biological functions. A complex network of intra-and intermolecular interactions, post-translational modifications and abundance levels is required to maintain the delicate balance of function essential for life. Subtle changes within this network can give rise to specific biological responses to environmental factors, onset of disease, normal aging, and other biological processes. Therefore, direct experimental observation of protein structures and interactions in relation to biological function is paramount to improved understanding of living systems.Chemical cross-linking has long been used as a method of fixation to preserve biological samples in the fields of histology and pathology (1). Protein interactions and topologies have also been studied with chemical cross-linking methods for many years (2-4). 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). 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. The resultant distance constraints are often used to refine crystal structure measurements and to assist de novo structure prediction with molecular modeling techniques (6, 7). Structural information derived through cross-linking experiments is largely complementary to structural information obtained through other techniques including hydrogen-deuterium exchange mass spectrometry, NMR, and x-ray cryst...

A New in Vivo Cross-linking Mass Spectrometry Platform to Define Protein–Protein Interactions in Living Cells

“…It is noted that the most abundant interactions identified in this work resulted from histones and structural proteins (supplemental Table S3), most likely attributed to their abundance as previously reported (24). In total, we identified 118 unique cross-linked peptides among the four (H2A, H2B, H3.2, and H4) core histones, with 47 from H2A-H2B, 13 from H2A-H3.2, 10 from H2B-H4, 8 from H2B-H3.2, 4 from H3.2-H4, and 1 from H2A-H4 pairs.…”

“…The latter provides direct molecular evidence describing the physical contacts between and within proteins (17). This information can be used for computational modeling to establish structural topologies of proteins and protein complexes (17)(18)(19)(20)(21)(22), as well as for generating experimentally derived protein interaction network topology maps (23,24). Thus, cross-linking mass spectrometry (XL-MS) strategies represent a powerful and emergent technology that possesses unparalleled capabilities for studying PPIs.…”

“…Despite their great potential, current XL-MS studies that have aimed to identify cross-linked peptides have been mostly limited to in vitro cross-linking experiments, with few successfully identifying protein interaction interfaces in living cells (24,25). This is largely because XL-MS studies remain challenging due to the inherent difficulty in the effective MS detection and accurate identification of cross-linked peptides, as well as in unambiguous assignment of cross-linked residues.…”

“…Various efforts have been made to address the limitations of XL-MS studies, resulting in new developments in bioinformatics tools for improved data interpretation (28 -32) and new designs of cross-linking reagents for enhanced MS analysis of cross-linked peptides (24,(33)(34)(35)(36)(37)(38)(39). Among these approaches, the development of new cross-linking reagents holds great promise for mapping PPIs on the systems level.…”

Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines

“…Using the protein interaction reporter concept based on gas phase cleavable, affinity tagged crosslinking reagents, Bruce and coworkers have shown applications to a diverse range of organisms such as Shewanella oneidensis [111], Escherichia coli [112 114], Pseudomonas aeruginosa [115], and even human cell lines [116]; other groups have reported similar concepts [117 119]. Recently, the group of Bruce also demonstrated that a quantitative dimension can be added to proteome wide crosslinking data with the help of metabolic stable isotope labeling [120].…”