Graphical Abstract

Protein-protein interactions (PPIs

Defining protein–protein interactions (PPIs) in their native environment is crucial to understanding protein structure and function. Cross-linking–mass spectrometry (XL-MS) has proven effective in capturing PPIs in living cells; however, the proteome coverage remains limited. Here, we have developed a robust in vivo XL-MS platform to facilitate in-depth PPI mapping by integrating a multifunctional MS-cleavable cross-linker with sample preparation strategies and high-resolution MS. The advancement of click chemistry–based enrichment significantly enhanced the detection of cross-linked peptides for proteome-wide analyses. This platform enabled the identification of 13,904 unique lysine–lysine linkages from in vivo cross-linked HEK 293 cells, permitting construction of the largest in vivo PPI network to date, comprising 6,439 interactions among 2,484 proteins. These results allowed us to generate a highly detailed yet panoramic portrait of human interactomes associated with diverse cellular pathways. The strategy presented here signifies a technological advancement for in vivo PPI mapping at the systems level and can be generalized for charting protein interaction landscapes in any organisms.

Cross-linking mass spectrometry (XL-MS) has become a powerful strategy for defining protein–protein interactions and elucidating architectures of large protein complexes. However, one of the inherent challenges in MS analysis of cross-linked peptides is their unambiguous identification. To facilitate this process, we have previously developed a series of amine-reactive sulfoxide-containing MS-cleavable cross-linkers. These MS-cleavable reagents have allowed us to establish a common robust XL-MS workflow that enables fast and accurate identification of cross-linked peptides using multistage tandem mass spectrometry (MSn). Although amine-reactive reagents targeting lysine residues have been successful, it remains difficult to characterize protein interaction interfaces with little or no lysine residues. To expand the coverage of protein interaction regions, we present here the development of a new acidic residue-targeting sulfoxide-containing MS-cleavable homobifunctional cross-linker, dihydrazide sulfoxide (DHSO). We demonstrate that DHSO cross-linked peptides display the same predictable and characteristic fragmentation pattern during collision induced dissociation as amine-reactive sulfoxide-containing MS-cleavable cross-linked peptides, thus permitting their simplified analysis and unambiguous identification by MSn. Additionally, we show that DHSO can provide complementary data to amine-reactive reagents. Collectively, this work not only enlarges the range of the application of XL-MS approaches but also further demonstrates the robustness and applicability of sulfoxide-based MS-cleavability in conjunction with various cross-linking chemistries.

The 26S proteasome is the macromolecular machine responsible for ATP/ubiquitin dependent degradation. As aberration in proteasomal degradation has been implicated in many human diseases, structural analysis of the human 26S proteasome complex is essential to advance our understanding of its action and regulation mechanisms. In recent years, cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for elucidating structural topologies of large protein assemblies, with its unique capability of studying protein complexes in cells. To facilitate the identification of cross-linked peptides, we have previously developed a robust amine reactive sulfoxide-containing MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). To better understand the structure and regulation of the human 26S proteasome, we have established new DSSO-based and XL-MS workflows by coupling with HB-tag based affinity purification to comprehensively examine protein-protein interactions within the 26S proteasome. In total, we have identified 447 unique lysine-to-lysine linkages delineating 67 interprotein and 26 intraprotein interactions, representing the largest cross-link dataset for proteasome complexes. In combination with EM maps and computational modeling, the architecture of the 26S proteasome was determined to infer its structural dynamics. In particular, three proteasome subunits Rpn1, Rpn6, and Rpt6 displayed multiple conformations that have not been previously reported. Additionally, cross-links between proteasome subunits and 15 proteasome interacting proteins including 9 known and 6 novel ones have been determined to demonstrate their physical interactions at the amino acid level. Our results have provided new insights on the dynamics of the 26S human proteasome and the methodologies presented here can be applied to study other protein complexes.

Oxidative stress has been implicated in multiple human neurological and other disorders. Proteasomes are multi-subunit proteases critical for the removal of oxidatively damaged proteins. To understand stress-associated human pathologies, it is important to uncover the molecular events underlying the regulation of proteasomes upon oxidative stress. To this end, we investigated HO stress-induced molecular changes of the human 26S proteasome and determined that stress-induced 26S proteasome disassembly is conserved from yeast to human. Moreover, we developed and employed a new proteomic approach, XAP ( cross-linking-assisted affinity purification), coupled with stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative MS, to capture and quantify several weakly bound proteasome-interacting proteins and examine their roles in stress-mediated proteasomal remodeling. Our results indicate that the adapter protein Ecm29 is the main proteasome-interacting protein responsible for stress-triggered remodeling of the 26S proteasome in human cells. Importantly, using a disuccinimidyl sulfoxide-based cross-linking MS platform, we mapped the interactions of Ecm29 within itself and with proteasome subunits and determined the architecture of the Ecm29-proteasome complex with integrative structure modeling. These results enabled us to propose a structural model in which Ecm29 intrudes on the interaction between the 20S core particle and the 19S regulatory particle in the 26S proteasome, disrupting the proteasome structure in response to oxidative stress.

Structural characterization of protein complexes is essential for the understanding of their function and regulation. However, it remains challenging due to limitations in existing tools. With recent technological improvements, cross-linking mass spectrometry (XL-MS) has become a powerful strategy to define protein–protein interactions and elucidate structural topologies of protein complexes. To further advance XL-MS studies, we present here the development of new isotope-coded MS-cleavable homobifunctional cross-linkers: d0- and d10-labeled dimethyl disuccinimidyl sulfoxide (DMDSSO). Detailed characterization of DMDSSO cross-linked peptides further demonstrates that sulfoxide-containing MS-cleavable cross-linkers offer robust and predictable MS2 fragmentation of cross-linked peptides, permitting subsequent MS3 analysis for simplified, unambiguous identification. Concurrent usage of these reagents provides a characteristic doublet pattern of DMDSSO cross-linked peptides, thus aiding in the confidence of cross-link identification by MSn analysis. More importantly, the unique isotopic profile permits quantitative analysis of cross-linked peptides and therefore expands the capability of XL-MS strategies to analyze both static and dynamic protein interactions. Together, our work has established a new XL-MS workflow for future studies toward the understanding of structural dynamics of protein complexes.

The COP9 signalosome (CSN) is an evolutionarily conserved eight-subunit (CSN1–8) protein complex that controls protein ubiquitination by deneddylating Cullin-RING E3 ligases (CRLs). The activation and function of CSN hinges on its structural dynamics, which has been challenging to decipher by conventional tools. Here, we have developed a multichemistry cross-linking mass spectrometry approach enabled by three mass spectometry-cleavable cross-linkers to generate highly reliable cross-link data. We applied this approach with integrative structure modeling to determine the interaction and structural dynamics of CSN with the recently discovered ninth subunit, CSN9, in solution. Our results determined the localization of CSN9 binding sites and revealed CSN9-dependent structural changes of CSN. Together with biochemical analysis, we propose a structural model in which CSN9 binding triggers CSN to adopt a configuration that facilitates CSN–CRL interactions, thereby augmenting CSN deneddylase activity. Our integrative structure analysis workflow can be generalized to define in-solution architectures of dynamic protein complexes that remain inaccessible to other approaches.