Protein Interaction Map of APOBEC3 Enzyme Family Reveals Deamination-Independent Role in Cellular Function.

Human APOBEC3 enzymes are a family of single-stranded (ss)DNA and RNA cytidine deaminases that act as part of the intrinsic immunity against viruses and retroelements. These enzymes deaminate cytosine to form uracil which can functionally inactivate or cause degradation of viral or retroelement genomes. In addition, APOBEC3s have deamination-independent antiviral activity through protein and nucleic acid interactions. If expression levels are misregulated, some APOBEC3 enzymes can access the human genome leading to deamination and mutagenesis, contributing to cancer initiation and evolution. While APOBEC3 enzymes are known to interact with large ribonucleoprotein complexes, the function and RNA dependence are not entirely understood. To further understand their cellular roles, we determined by affinity purification mass spectrometry (AP-MS) the protein interaction network for the human APOBEC3 enzymes and mapped a diverse set of protein-protein and protein-RNA mediated interactions. Our analysis identified novel RNA-mediated interactions between APOBEC3C, APOBEC3H Haplotype I and II, and APOBEC3G with spliceosome proteins, and APOBEC3G and APOBEC3H Haplotype I with proteins involved in tRNA methylation and ncRNA export from the nucleus. In addition, we identified RNA-independent protein-protein interactions with APOBEC3B, APOBEC3D, and APOBEC3F and the prefoldin family of protein-folding chaperones. Interaction between prefoldin 5 (PFD5) and APOBEC3B disrupted the ability of PFD5 to induce degradation of the oncogene cMyc, implicating the APOBEC3B protein interaction network in cancer. Altogether, the results uncover novel functions and interactions of the APOBEC3 family and suggest they may have fundamental roles in cellular RNA biology, their protein-protein interactions are not redundant, and there are protein-protein interactions with tumor suppressors, suggesting a role in cancer biology. Data are available via ProteomeXchange with the identifier PXD044275.

CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells.

During HIV infection of CD4+ T cells, ubiquitin pathways are essential to viral replication and host innate immune response; however, the role of specific E3 ubiquitin ligases is not well understood. Proteomics analyses identified 116 single-subunit E3 ubiquitin ligases expressed in activated primary human CD4+ T cells. Using a CRISPR-based arrayed spreading infectivity assay, we systematically knocked out 116 E3s from activated primary CD4+ T cells and infected them with NL4-3 GFP reporter HIV-1. We found 10 E3s significantly positively or negatively affected HIV infection in activated primary CD4+ T cells, including UHRF1 (pro-viral) and TRAF2 (anti-viral). Furthermore, deletion of either TRAF2 or UHRF1 in three JLat models of latency spontaneously increased HIV transcription. To verify this effect, we developed a CRISPR-compatible resting primary human CD4+ T cell model of latency. Using this system, we found that deletion of TRAF2 or UHRF1 initiated latency reactivation and increased virus production from primary human resting CD4+ T cells, suggesting these two E3s represent promising targets for future HIV latency reversal strategies. IMPORTANCE: HIV, the virus that causes AIDS, heavily relies on the machinery of human cells to infect and replicate. Our study focuses on the host cell's ubiquitination system which is crucial for numerous cellular processes. Many pathogens, including HIV, exploit this system to enhance their own replication and survival. E3 proteins are part of the ubiquitination pathway that are useful drug targets for host-directed therapies. We interrogated the 116 E3s found in human immune cells known as CD4+ T cells, since these are the target cells infected by HIV. Using CRISPR, a gene-editing tool, we individually removed each of these enzymes and observed the impact on HIV infection in human CD4+ T cells isolated from healthy donors. We discovered that 10 of the E3 enzymes had a significant effect on HIV infection. Two of them, TRAF2 and UHRF1, modulated HIV activity within the cells and triggered an increased release of HIV from previously dormant or "latent" cells in a new primary T cell assay. This finding could guide strategies to perturb hidden HIV reservoirs, a major hurdle to curing HIV. Our study offers insights into HIV-host interactions, identifies new factors that influence HIV infection in immune cells, and introduces a novel methodology for studying HIV infection and latency in human immune cells.

Protein interaction map of APOBEC3 enzyme family reveals deamination-independent role in cellular function.

Human APOBEC3 enzymes are a family of single-stranded (ss)DNA and RNA cytidine deaminases that act as part of the intrinsic immunity against viruses and retroelements. These enzymes deaminate cytosine to form uracil which can functionally inactivate or cause degradation of viral or retroelement genomes. In addition, APOBEC3s have deamination independent antiviral activity through protein and nucleic acid interactions. If expression levels are misregulated, some APOBEC3 enzymes can access the human genome leading to deamination and mutagenesis, contributing to cancer initiation and evolution. While APOBEC3 enzymes are known to interact with large ribonucleoprotein complexes, the function and RNA dependence is not entirely understood. To further understand their cellular roles, we determined by affinity purification mass spectrometry (AP-MS) the protein interaction network for the human APOBEC3 enzymes and map a diverse set of protein-protein and protein-RNA mediated interactions. Our analysis identified novel RNA-mediated interactions between APOBEC3C, APOBEC3H Haplotype I and II, and APOBEC3G with spliceosome proteins, and APOBEC3G and APOBEC3H Haplotype I with proteins involved in tRNA methylation and ncRNA export from the nucleus. In addition, we identified RNA-independent protein-protein interactions with APOBEC3B, APOBEC3D, and APOBEC3F and the prefoldin family of protein folding chaperones. Interaction between prefoldin 5 (PFD5) and APOBEC3B disrupted the ability of PFD5 to induce degradation of the oncogene cMyc, implicating the APOBEC3B protein interaction network in cancer. Altogether, the results uncover novel functions and interactions of the APOBEC3 family and suggest they may have fundamental roles in cellular RNA biology, their protein-protein interactions are not redundant, and there are protein-protein interactions with tumor suppressors, suggesting a role in cancer biology.

De novo protein identification in mammalian sperm using in situ cryoelectron tomography and AlphaFold2 docking.

To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine their structures in vitro. Here, we combined cellular cryoelectron tomography (cryo-ET) and AlphaFold2 modeling to address these questions and understand how mammalian sperm are built in situ. Our cellular cryo-ET and subtomogram averaging provided 6.0-Å reconstructions of axonemal microtubule structures. The well-resolved tertiary structures allowed us to unbiasedly match sperm-specific densities with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105, and SPACA9 as novel microtubule-associated proteins. These proteins form an extensive interaction network crosslinking the lumen of axonemal doublet microtubules, suggesting their roles in modulating the mechanical properties of the filaments. Indeed, Tekt5 -/- sperm possess more deformed flagella with 180° bends. Together, our studies presented a cellular visual proteomics workflow and shed light on the in vivo functions of Tektin 5.

Structure and dynamics of the essential endogenous mycobacterial polyketide synthase Pks13.

The mycolic acid layer of the Mycobacterium tuberculosis cell wall is essential for viability and virulence, and the enzymes responsible for its synthesis are targets for antimycobacterial drug development. Polyketide synthase 13 (Pks13) is a module encoding several enzymatic and transport functions that carries out the condensation of two different long-chain fatty acids to produce mycolic acids. We determined structures by cryogenic-electron microscopy of dimeric multi-enzyme Pks13 purified from mycobacteria under normal growth conditions, captured with native substrates. Structures define the ketosynthase (KS), linker and acyl transferase (AT) domains at 1.8 Å resolution and two alternative locations of the N-terminal acyl carrier protein. These structures suggest intermediate states on the pathway for substrate delivery to the KS domain. Other domains, visible at lower resolution, are flexible relative to the KS-AT core. The chemical structures of three bound endogenous long-chain fatty acid substrates were determined by electrospray ionization mass spectrometry.

Structure and dynamics of the essential endogenous mycobacterial polyketide synthase Pks13.

Mycobacterium tuberculosis is currently the leading cause of death by any bacterial infection1. The mycolic acid layer of the cell wall is essential for viability and virulence, and the enzymes responsible for its synthesis are therefore front line targets for antimycobacterial drug development2,3. Polyketide synthase 13 (Pks13) is a module comprised of a closely symmetric parallel dimer of chains, each encoding several enzymatic and transport functions, that carries out the condensation of two different very long chain fatty acids to produce mycolic acids that are essential components of the mycobacterial cell wall. Consequently individual enzymatic domains of Pks13 are targets for antimycobacterial drug development4. To understand this machinery, we sought to determine the structure and domain trajectories of the dimeric multi-enzyme Pks13, a 2×198,426 Dalton complex, from protein purified endogenously from mycobacteria under normal growth conditions, to capture it with normal substrates bound trapped 'in action'. Structures of the multi-domain assembly revealed by cryogenic electron microscopy (cryoEM) define the ketosynthase (KS), linker, and acyltransferase (AT) domains, each at atomic resolution (1.8Å), with bound substrates defined at 2.4Å and 2.9Å resolution. Image classification reveals two distinct structures with alternate locations of the N-terminal acyl carrier protein (termed ACP1a, ACP1b) seen at 3.6Å and 4.6Å resolution respectively. These two structures suggest plausible intermediate states, related by a ~60Å movement of ACP1, on the pathway for substrate delivery from the fatty acyl-ACP ligase (FadD32) to the ketosynthase domain. The linking sequence between ACP1 and the KS includes an 11 amino acid sequence with 6 negatively charged side chains that lies in different positively charged grooves on the KS in ACP1a versus ACP1b structures. This charge complementarity between the extended chain and the grooves suggests some stabilization of these two distinct orientations. Other domains are visible at lower resolution and indicate flexibility relative to the KS-AT core. The chemical structures of three bound endogenous long chain fatty acid substrates with their proximal regions defined in the structures were determined by electrospray ionization mass spectrometry. The domain proximities were probed by chemical cross-linking and identified by mass spectrometry. These were incorporated into integrative structure modeling to define multiple domain configurations that transport the very long fatty acid chains throughout the multistep Pks13 mediated synthetic pathway.

From systems to structure - using genetic data to model protein structures.

Understanding the effects of genetic variation is a fundamental problem in biology that requires methods to analyse both physical and functional consequences of sequence changes at systems-wide and mechanistic scales. To achieve a systems view, protein interaction networks map which proteins physically interact, while genetic interaction networks inform on the phenotypic consequences of perturbing these protein interactions. Until recently, understanding the molecular mechanisms that underlie these interactions often required biophysical methods to determine the structures of the proteins involved. The past decade has seen the emergence of new approaches based on coevolution, deep mutational scanning and genome-scale genetic or chemical-genetic interaction mapping that enable modelling of the structures of individual proteins or protein complexes. Here, we review the emerging use of large-scale genetic datasets and deep learning approaches to model protein structures and their interactions, and discuss the integration of structural data from different sources.

Protein interaction landscapes revealed by advanced in vivo cross-linking-mass spectrometry.

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.

Characterization of an A3G-VifHIV-1-CRL5-CBFß Structure Using a Cross-linking Mass Spectrometry Pipeline for Integrative Modeling of Host-Pathogen Complexes.

Structural analysis of host-pathogen protein complexes remains challenging, largely due to their structural heterogeneity. Here, we describe a pipeline for the structural characterization of these complexes using integrative structure modeling based on chemical cross-links and residue-protein contacts inferred from mutagenesis studies. We used this approach on the HIV-1 Vif protein bound to restriction factor APOBEC3G (A3G), the Cullin-5 E3 ring ligase (CRL5), and the cellular transcription factor Core Binding Factor Beta (CBFß) to determine the structure of the (A3G-Vif-CRL5-CBFß) complex. Using the MS-cleavable DSSO cross-linker to obtain a set of 132 cross-links within this reconstituted complex along with the atomic structures of the subunits and mutagenesis data, we computed an integrative structure model of the heptameric A3G-Vif-CRL5-CBFß complex. The structure, which was validated using a series of tests, reveals that A3G is bound to Vif mostly through its N-terminal domain. Moreover, the model ensemble quantifies the dynamic heterogeneity of the A3G C-terminal domain and Cul5 positions. Finally, the model was used to rationalize previous structural, mutagenesis and functional data not used for modeling, including information related to the A3G-bound and unbound structures as well as mapping functional mutations to the A3G-Vif interface. The experimental and computational approach described here is generally applicable to other challenging host-pathogen protein complexes.

Proteomic Approaches to Study SARS-CoV-2 Biology and COVID-19 Pathology.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), was declared a pandemic infection in March 2020. As of December 2020, two COVID-19 vaccines have been authorized for emergency use by the U.S. Food and Drug Administration, but there are no effective drugs to treat COVID-19, and pandemic mitigation efforts like physical distancing have had acute social and economic consequences. In this perspective, we discuss how the proteomic research community can leverage technologies and expertise to address the pandemic by investigating four key areas of study in SARS-CoV-2 biology. Specifically, we discuss how (1) mass spectrometry-based structural techniques can overcome limitations and complement traditional structural approaches to inform the dynamic structure of SARS-CoV-2 proteins, complexes, and virions; (2) virus-host protein-protein interaction mapping can identify the cellular machinery required for SARS-CoV-2 replication; (3) global protein abundance and post-translational modification profiling can characterize signaling pathways that are rewired during infection; and (4) proteomic technologies can aid in biomarker identification, diagnostics, and drug development in order to monitor COVID-19 pathology and investigate treatment strategies. Systems-level high-throughput capabilities of proteomic technologies can yield important insights into SARS-CoV-2 biology that are urgently needed during the pandemic, and more broadly, can inform coronavirus virology and host biology.

Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a grave threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analyses for all three viruses. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 ORF9b, an interaction we structurally characterized using cryo-electron microscopy. Combining genetically validated host factors with both COVID-19 patient genetic data and medical billing records identified molecular mechanisms and potential drug treatments that merit further molecular and clinical study.

The Global Phosphorylation Landscape of SARS-CoV-2 Infection.

The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.

Structural basis of CD4 downregulation by HIV-1 Nef.

The HIV-1 Nef protein suppresses multiple immune surveillance mechanisms to promote viral pathogenesis and is an attractive target for the development of novel therapeutics. A key function of Nef is to remove the CD4 receptor from the cell surface by hijacking clathrin- and adaptor protein complex 2 (AP2)-dependent endocytosis. However, exactly how Nef does this has been elusive. Here, we describe the underlying mechanism as revealed by a 3.0-Å crystal structure of a fusion protein comprising Nef and the cytoplasmic domain of CD4 bound to the tetrameric AP2 complex. An intricate combination of conformational changes occurs in both Nef and AP2 to enable CD4 binding and downregulation. A pocket on Nef previously identified as crucial for recruiting class I MHC is also responsible for recruiting CD4, revealing a potential approach to inhibit two of Nef's activities and sensitize the virus to immune clearance.

A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing.

An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 290,000 people since the end of 2019, killed over 12,000, and caused worldwide social and economic disruption 1,2 . There are currently no antiviral drugs with proven efficacy nor are there vaccines for its prevention. Unfortunately, the scientific community has little knowledge of the molecular details of SARS-CoV-2 infection. To illuminate this, we cloned, tagged and expressed 26 of the 29 viral proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), which identified 332 high confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 existing FDA-approved drugs, drugs in clinical trials and/or preclinical compounds, that we are currently evaluating for efficacy in live SARS-CoV-2 infection assays. The identification of host dependency factors mediating virus infection may provide key insights into effective molecular targets for developing broadly acting antiviral therapeutics against SARS-CoV-2 and other deadly coronavirus strains.

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.

A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein-protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.

A systems approach to infectious disease.

Ongoing social, political and ecological changes in the 21st century have placed more people at risk of life-threatening acute and chronic infections than ever before. The development of new diagnostic, prophylactic, therapeutic and curative strategies is critical to address this burden but is predicated on a detailed understanding of the immensely complex relationship between pathogens and their hosts. Traditional, reductionist approaches to investigate this dynamic often lack the scale and/or scope to faithfully model the dual and co-dependent nature of this relationship, limiting the success of translational efforts. With recent advances in large-scale, quantitative omics methods as well as in integrative analytical strategies, systems biology approaches for the study of infectious disease are quickly forming a new paradigm for how we understand and model host-pathogen relationships for translational applications. Here, we delineate a framework for a systems biology approach to infectious disease in three parts: discovery - the design, collection and analysis of omics data; representation - the iterative modelling, integration and visualization of complex data sets; and application - the interpretation and hypothesis-based inquiry towards translational outcomes.

EpCAM homo-oligomerization is not the basis for its role in cell-cell adhesion.

Cell-surface tumor marker EpCAM plays a key role in proliferation, differentiation and adhesion processes in stem and epithelial cells. It is established as a cell-cell adhesion molecule, forming intercellular interactions through homophilic association. However, the mechanism by which such interactions arise has not yet been fully elucidated. Here, we first show that EpCAM monomers do not associate into oligomers that would resemble an inter-cellular homo-oligomer, capable of mediating cell-cell adhesion, by using SAXS, XL-MS and bead aggregation assays. Second, we also show that EpCAM forms stable dimers on the surface of a cell with pre-formed cell-cell contacts using FLIM-FRET; however, no inter-cellular homo-oligomers were detectable. Thus, our study provides clear evidence that EpCAM indeed does not function as a homophilic cell adhesion molecule and therefore calls for a significant revision of its role in both normal and cancerous tissues. In the light of this, we strongly support the previously suggested name Epithelial Cell Activating Molecule instead of the Epithelial Cell Adhesion Molecule.

A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress.

Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism. However, heterozygous knockout of the murine BAG3 gene did not cause a detectable phenotype. To model BAG3 cardiomyopathy in a human system, we generated an isogenic series of human induced pluripotent stem cells (iPSCs) with loss-of-function mutations in BAG3. Heterozygous BAG3 mutations reduced protein expression, disrupted myofibril structure, and compromised contractile function in iPSC-derived cardiomyocytes (iPS-CMs). BAG3-deficient iPS-CMs were particularly sensitive to further myofibril disruption and contractile dysfunction upon exposure to proteasome inhibitors known to cause cardiotoxicity. We performed affinity tagging of the endogenous BAG3 protein and mass spectrometry proteomics to further define the cardioprotective chaperone complex that BAG3 coordinates in the human heart. Our results establish a model for evaluating protein quality control pathways in human cardiomyocytes and their potential as therapeutic targets and susceptibility factors for cardiac drug toxicity.

The RNA Exosome Syncs IAV-RNAPII Transcription to Promote Viral Ribogenesis and Infectivity.

The nuclear RNA exosome is an essential multi-subunit complex that controls RNA homeostasis. Congenital mutations in RNA exosome genes are associated with neurodegenerative diseases. Little is known about the role of the RNA exosome in the cellular response to pathogens. Here, using NGS and human and mouse genetics, we show that influenza A virus (IAV) ribogenesis and growth are suppressed by impaired RNA exosome activity. Mechanistically, the nuclear RNA exosome coordinates the initial steps of viral transcription with RNAPII at host promoters. The viral polymerase complex co-opts the nuclear RNA exosome complex and cellular RNAs en route to 3' end degradation. Exosome deficiency uncouples chromatin targeting of the viral polymerase complex and the formation of cellular:viral RNA hybrids, which are essential RNA intermediates that license transcription of antisense genomic viral RNAs. Our results suggest that evolutionary arms races have shaped the cellular RNA quality control machinery.

Synthesis of two new enrichable and MS-cleavable cross-linkers to define protein-protein interactions by mass spectrometry.

The cross-linking Mass Spectrometry (XL-MS) technique extracts structural information from protein complexes without requiring highly purified samples, crystallinity, or large amounts of material. However, there are challenges to applying the technique to protein complexes in vitro, and those challenges become more daunting with in vivo experiments. Issues include effective detection and identification of cross-linked peptides from complex mixtures. While MS-cleavable cross-linkers facilitate the sequencing and identification of cross-linked peptides, enrichable cross-linkers increase their detectability by allowing their separation from non-cross-linked peptides prior to MS analysis. Although a number of cross-linkers with single functionality have been developed in recent years, an ideal reagent would incorporate both capabilities for XL-MS studies. Therefore, two new cross-linkers have been designed and prepared that incorporate an azide (azide-A-DSBSO) or alkyne (alkyne-A-DSBSO) to enable affinity purification strategies based on click chemistry. The integration of an acid cleavage site next to the enrichment handle allows easy recovery of cross-linked products during affinity purification. In addition, these sulfoxide containing cross-linking reagents possess robust MS-cleavable bonds to facilitate fast and easy identification of cross-linked peptides using MS analysis. Optimized, gram-scale syntheses of these cross-linkers have been developed and the azide-A-DSBSO cross-linker has been evaluated with peptides and proteins to demonstrate its utility in XL-MS analysis.