Proteomic profiling of bronchoalveolar lavage fluid uncovers protein clusters linked to survival in idiopathic forms of interstitial lung disease.

Background: Idiopathic interstitial pneumonias (IIPs) such as idiopathic pulmonary fibrosis (IPF) and interstitial pneumonia with autoimmune features (IPAF), present diagnostic and therapeutic challenges due to their heterogeneous nature. This study aimed to identify intrinsic molecular signatures within the lung microenvironment of these IIPs through proteomic analysis of bronchoalveolar lavage fluid (BALF). Methods: Patients with IIP (n=23) underwent comprehensive clinical evaluation including pre-treatment bronchoscopy and were compared to controls without lung disease (n=5). Proteomic profiling of BALF was conducted using label-free quantitative methods. Unsupervised cluster analyses identified protein expression profiles which were then analyzed to predict survival outcomes and investigate associated pathways. Results: Proteomic profiling successfully differentiated IIP from controls. k-means clustering, based on protein expression revealed three distinct IIP clusters, which were not associated with age, smoking history, or baseline pulmonary function. These clusters had unique survival trajectories and provided more accurate survival predictions than the Gender Age Physiology (GAP) index (C-index 0.794 vs. 0.709). The cluster with the worst prognosis featured decreased inflammatory signaling and complement activation, with pathway analysis highlighting altered immune response pathways related to immunoglobulin production and B cell-mediated immunity. Conclusions: The unsupervised clustering of BALF proteomics provided a novel stratification of IIP patients, with potential implications for prognostic and therapeutic targeting. The identified molecular phenotypes underscore the diversity within the IIP classification and the potential importance of personalized treatments for these conditions. Future validation in larger, multi-ethnic cohorts is essential to confirm these findings and to explore their utility in clinical decision-making for patients with IIP.

DCLK1-Mediated Regulation of Invadopodia Dynamics and Matrix Metalloproteinase Trafficking Drives Invasive Progression in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a major health concern due to its high mortality from poor treatment responses and locoregional tumor invasion into life sustaining structures in the head and neck. A deeper comprehension of HNSCC invasion mechanisms holds the potential to inform targeted therapies that may enhance patient survival. We previously reported that doublecortin like kinase 1 (DCLK1) regulates invasion of HNSCC cells. Here, we tested the hypothesis that DCLK1 regulates proteins within invadopodia to facilitate HNSCC invasion. Invadopodia are specialized subcellular protrusions secreting matrix metalloproteinases that degrade the extracellular matrix (ECM). Through a comprehensive proteome analysis comparing DCLK1 control and shDCLK1 conditions, our findings reveal that DCLK1 plays a pivotal role in regulating proteins that orchestrate cytoskeletal and ECM remodeling, contributing to cell invasion. Further, we demonstrate in TCGA datasets that DCLK1 levels correlate with increasing histological grade and lymph node metastasis. We identified higher expression of DCLK1 in the leading edge of HNSCC tissue. Knockdown of DCLK1 in HNSCC reduced the number of invadopodia, cell adhesion and colony formation. Using super resolution microscopy, we demonstrate localization of DCLK1 in invadopodia and colocalization with mature invadopodia markers TKS4, TKS5, cortactin and MT1-MMP. We carried out phosphoproteomics and validated using immunofluorescence and proximity ligation assays, the interaction between DCLK1 and motor protein KIF16B. Pharmacological inhibition or knockdown of DCLK1 reduced interaction with KIF16B, secretion of MMPs, and cell invasion. This research unveils a novel function of DCLK1 within invadopodia to regulate the trafficking of matrix degrading cargo. The work highlights the impact of targeting DCLK1 to inhibit locoregional invasion, a life-threatening attribute of HNSCC.

Distinct regions within SAP25 recruit O-linked glycosylation, DNA demethylation, and ubiquitin ligase and hydrolase activities to the Sin3/HDAC complex.

Epigenetic control of gene expression is crucial for maintaining gene regulation. Sin3 is an evolutionarily conserved repressor protein complex mainly associated with histone deacetylase (HDAC) activity. A large number of proteins are part of Sin3/HDAC complexes, and the function of most of these members remains poorly understood. SAP25, a previously identified Sin3A associated protein of 25 kDa, has been proposed to participate in regulating gene expression programs involved in the immune response but the exact mechanism of this regulation is unclear. SAP25 is not expressed in HEK293 cells, which hence serve as a natural knockout system to decipher the molecular functions uniquely carried out by this Sin3/HDAC subunit. Using molecular, proteomic, protein engineering, and interaction network approaches, we show that SAP25 interacts with distinct enzymatic and regulatory protein complexes in addition to Sin3/HDAC. While the O-GlcNAc transferase (OGT) and the TET1 /TET2/TET3 methylcytosine dioxygenases have been previously linked to Sin3/HDAC, in HEK293 cells, these interactions were only observed in the affinity purification in which an exogenously expressed SAP25 was the bait. Additional proteins uniquely recovered from the Halo-SAP25 pull-downs included the SCF E3 ubiquitin ligase complex SKP1/FBXO3/CUL1 and the ubiquitin carboxyl-terminal hydrolase 11 (USP11), which have not been previously associated with Sin3/HDAC. Finally, we use mutational analysis to demonstrate that distinct regions of SAP25 participate in its interaction with USP11, OGT/TETs, and SCF(FBXO3).) These results suggest that SAP25 may function as an adaptor protein to coordinate the assembly of different enzymatic complexes to control Sin3/HDAC-mediated gene expression.

Neutrophil extracellular traps linked to idiopathic pulmonary fibrosis severity and survival.

Background: Idiopathic pulmonary fibrosis (IPF) leads to progressive loss of lung function and mortality. Understanding mechanisms and markers of lung injury in IPF is paramount to improving outcomes for these patients. Despite the lack of systemic involvement in IPF, many analyses focus on identifying circulating prognostic markers. Using a proteomic discovery method followed by ELISA validation in multiple IPF lung compartments and cohorts we explored novel markers of IPF survival. Methods: In our discovery analysis, agnostic label-free quantitative proteomics differentiated lung tissue protein expression based on survival trajectory (n=10). Following selection of the candidate pathway (neutrophil extracellular trap (NET) formation), we subsequently validated the presence of NETs in the IPF lung microenvironment using fully quantitative assays of known NET remnants in separate IPF cohorts (n=156 and n=52) with bronchoalveolar lavage fluid. We then assessed the correlation of these markers with baseline pulmonary function and survival. Results: Discovery lung tissue proteomics identified NET formation as significantly associated with poor IPF survival. Using fully quantitative confirmatory tests for reproducibility we confirmed the presence of NET markers in IPF BALF and found significant correlations with worse pulmonary function in both cohorts (p<0.03 and p = 0.04 respectively). In the survival cohort, higher levels of NET markers predicted worse survival after adjusting for gender, age, and baseline physiologic severity (hazard ratio range: 1.79-2.19). Conclusions: NET markers were associated with disease severity and worse survival in IPF. These findings suggest NET formation contributes to lung injury and decreased survival in IPF and may represent a potential therapeutic target.

The antiproliferative effect of FGF2 in K-Ras-driven tumor cells involves modulation of rRNA and the nucleolus.

The nucleolus is sensitive to stress and can orchestrate a chain of cellular events in response to stress signals. Despite being a growth factor, FGF2 has antiproliferative and tumor-suppressive functions in some cellular contexts. In this work, we investigated how the antiproliferative effect of FGF2 modulates chromatin-, nucleolus- and rDNA-associated proteins. The chromatin and nucleolar proteome indicated that FGF2 stimulation modulates proteins related to transcription, rRNA expression and chromatin-remodeling proteins. The global transcriptional rate and nucleolus area increased along with nucleolar disorganization upon 24 h of FGF2 stimulation. FGF2 stimulation induced immature rRNA accumulation by increasing rRNA transcription. The rDNA-associated protein analysis reinforced that FGF2 stimulus interferes with transcription and rRNA processing. RNA Pol I inhibition partially reversed the growth arrest induced by FGF2, indicating that changes in rRNA expression might be crucial for triggering the antiproliferative effect. Taken together, we demonstrate that the antiproliferative FGF2 stimulus triggers significant transcriptional changes and modulates the main cell transcription site, the nucleolus.

Acoustic diffraction-resistant adaptive profile technology (ADAPT) for elasticity imaging.

Acoustic beam shaping with high degrees of freedom is critical for applications such as ultrasound imaging, acoustic manipulation, and stimulation. However, the ability to fully control the acoustic pressure profile over its propagation path has not yet been achieved. Here, we demonstrate an acoustic diffraction-resistant adaptive profile technology (ADAPT) that can generate a propagation-invariant beam with an arbitrarily desired profile. By leveraging wave number modulation and beam multiplexing, we develop a general framework for creating a highly flexible acoustic beam with a linear array ultrasonic transducer. The designed acoustic beam can also maintain the beam profile in lossy material by compensating for attenuation. We show that shear wave elasticity imaging is an important modality that can benefit from ADAPT for evaluating tissue mechanical properties. Together, ADAPT overcomes the existing limitation of acoustic beam shaping and can be applied to various fields, such as medicine, biology, and material science.

Small Intestinal Polyp Burden in Pediatric Peutz-Jeghers Syndrome Assessed through Capsule Endoscopy: A Longitudinal Study.

The management of pediatric Peutz-Jeghers Syndrome (PJS) focuses on the prevention of intussusception complicating small intestinal (SI) polyposis. This hinges on the accurate appraisal of the polyp burden to tailor therapeutic interventions. Video Capsule Endoscopy (VCE) is an established tool to study SI polyps in children, but an in-depth characterization of polyp burden in this population is lacking. Methods: We performed a retrospective longitudinal cross-sectional analysis of VCE studies in pediatric PJS patients at our institution (CMKC) from 2010 to 2020. Demographic, clinical, and VCE findings reported by three reviewers in tandem were accrued. Polyp burden variables were modeled as functions of patient and study characteristics using linear mixed models adjusted for clustering. Results: The cohort included 15 patients. The total small bowel polyp count and largest polyp size clustered under 30 polyps and <20 mm in size. Luminal occlusion correlated closely with the estimated polyp size. Polyp distribution favored proximal (77%) over distal (66%) small bowel involvement. The adjusted largest polyp size was greater in males. Double Balloon Enteroscopy was associated with a decreased polyp burden. Conclusions: The polyp burden in pediatric PJS patients favors the proximal third of the small intestine, with relatively small numbers and a polyp size amenable to resection through enteroscopy. Male gender and older age were related to an increased polyp burden.

Serial Capture Affinity Purification and Integrated Structural Modeling of the H3K4me3 Binding and DNA Damage Related WDR76:SPIN1 Complex.

WDR76 is a multifunctional protein involved in many cellular functions. With a diverse and complicated protein interaction network, dissecting the structure and function of specific WDR76 complexes is needed. We previously demonstrated the ability of the Serial Capture Affinity Purification (SCAP) method to isolate specific complexes by introducing two proteins of interest as baits at the same time. Here, we applied SCAP to dissect a subpopulation of WDR76 in complex with SPIN1, a histone marker reader that specifically recognizes trimethylated histone H3 lysine4 (H3K4me3). In contrast to the SCAP analysis of the SPIN1:SPINDOC complex, H3K4me3 was copurified with the WDR76:SPIN1 complex. In combination with crosslinking mass spectrometry, we built an integrated structural model of the complex which revealed that SPIN1 recognized the H3K4me3 epigenetic mark while interacting with WDR76. Lastly, interaction network analysis of copurifying proteins revealed the potential role of the WDR76:SPIN1 complex in the DNA damage response. Teaser: In contrast to the SPINDOC/SPIN1 complex, analyses reveal that the WDR76/SPIN1 complex interacts with core histones and is involved in DNA damage.

Manufacturing-dependent change in biological activity of the TLR4 agonist GSK1795091 and implications for lipid A analog development.

A phase I trial (NCT03447314; 204686) evaluated the safety and efficacy of GSK1795091, a Toll-like receptor 4 (TLR4) agonist, in combination with immunotherapy (GSK3174998 [anti-OX40 monoclonal antibody], GSK3359609 [anti-ICOS monoclonal antibody], or pembrolizumab) in patients with solid tumors. The primary endpoint was safety; other endpoints included efficacy, pharmacokinetics, and pharmacodynamics (PD). Manufacturing of GSK1795091 formulation was modified during the trial to streamline production and administration, resulting in reduced PD (cytokine) activity. Fifty-four patients received GSK1795091 with a combination partner; 32 received only the modified GSK1795091 formulation, 15 received only the original formulation, and seven switched mid-study from the original to the modified formulation. Despite the modified formulation demonstrating higher systemic GSK1795091 exposure compared with the original formulation, the transient, dose-dependent elevations in cytokine and chemokine concentrations were no longer observed (e.g., IP-10, IL10, IL1-RA). Most patients (51/54; 94%) experienced ≥1 treatment-emergent adverse event (TEAE) during the study. Safety profiles were similar between formulations, but a higher incidence of TEAEs associated with immune responses (chills, fatigue, pyrexia, nausea, and vomiting) were observed with the original formulation. No conclusions can be made regarding GSK1795091 anti-tumor activity due to the limited data collected. Manufacturing changes were hypothesized to have caused the change in biological activity in this study. Structural characterization revealed GSK1795091 aggregate size in the modified formulation to be twice that in the original formulation, suggesting a negative correlation between GSK1795091 aggregate size and PD activity. This may have important clinical implications for future development of structurally similar compounds.

Acute exercise dynamically modulates the hepatic mitochondrial proteome.

Exercise powerfully increases energy metabolism and substrate flux in tissues, a process reliant on dramatic changes in mitochondrial energetics. Liver mitochondria play a multi-factorial role during exercise to fuel hepatic glucose output. We previously showed acute exercise activates hepatic mitophagy, a pathway to recycle low-functioning/damaged mitochondria, however little is known how individual bouts of exercise alters the hepatic mitochondrial proteome. Here we leveraged proteomics to examine changes in isolated hepatic mitochondria both immediately after and 2 hours post an acute, 1 hour bout of treadmill exercise in female mice. Further, we utilized leupeptin, a lysosomal inhibitor, to capture and measure exercise-induced changes in mitochondrial proteins that would have been unmeasured due to their targeting for lysosomal degradation. Proteomic analysis of enriched hepatic mitochondria identified 3241 total proteins. Functional enrichment analysis revealed robust enrichment for proteins critical to the mitochondria including metabolic pathways, tricarboxylic acid cycle, and electron transport system. Compared to the sedentary condition, exercise elevated processes regulating lipid localization, Il-5 signaling, and protein phosphorylation in isolated mitochondria. t-SNE analysis identified 4 unique expressional clusters driven by time-dependent changes in protein expression. Isolation of proteins significantly altered with exercise from each cluster revealed influences of leupeptin and exercise both independently and cooperatively modulating mitochondrial protein expressional profiles. Overall, we provide evidence that acute exercise rapidly modulates changes in the proteins/pathways of isolated hepatic mitochondria that include fatty acid metabolism/storage, post-translational protein modification, inflammation, and oxidative stress. In conclusion, the hepatic mitochondrial proteome undergoes extensive remodeling with a bout of exercise.

Cytidine acetylation yields a hypoinflammatory synthetic messenger RNA.

Synthetic messenger RNA (mRNA) is an emerging therapeutic platform with important applications in oncology and infectious disease. Effective mRNA medicines must be translated by the ribosome but not trigger a strong nucleic acid-mediated immune response. To expand the medicinal chemistry toolbox for these agents, here we report the properties of the naturally occurring nucleobase N4-acetylcytidine (ac4C) in synthetic mRNAs. We find that ac4C is compatible with, but does not enhance, protein production in the context of synthetic mRNA reporters. However, replacement of cytidine with ac4C diminishes inflammatory gene expression in immune cells caused by synthetic mRNAs. Chemoproteomic capture indicates that ac4C alters the protein interactome of synthetic mRNAs, reducing binding to cytidine-binding proteins and an immune sensor. Overall, our studies illustrate the unique ability of ac4C to modulate RNA-protein interactions and provide a foundation for using N4-cytidine acylation to fine-tune the properties of nucleic acid therapeutics.

Structural basis of the interaction between SETD2 methyltransferase and hnRNP L paralogs for governing co-transcriptional splicing.

The RNA recognition motif (RRM) binds to nucleic acids as well as proteins. More than one such domain is found in the pre-mRNA processing hnRNP proteins. While the mode of RNA recognition by RRMs is known, the molecular basis of their protein interaction remains obscure. Here we describe the mode of interaction between hnRNP L and LL with the methyltransferase SETD2. We demonstrate that for the interaction to occur, a leucine pair within a highly conserved stretch of SETD2 insert their side chains in hydrophobic pockets formed by hnRNP L RRM2. Notably, the structure also highlights that RRM2 can form a ternary complex with SETD2 and RNA. Remarkably, mutating the leucine pair in SETD2 also results in its reduced interaction with other hnRNPs. Importantly, the similarity that the mode of SETD2-hnRNP L interaction shares with other related protein-protein interactions reveals a conserved design by which splicing regulators interact with one another.

Perturbation of BRMS1 interactome reveals pathways that impact metastasis.

Breast Cancer Metastasis Suppressor 1 (BRMS1) expression is associated with longer patient survival in multiple cancer types. Understanding BRMS1 functionality will provide insights into both mechanism of action and will enhance potential therapeutic development. In this study, we confirmed that the C-terminus of BRMS1 is critical for metastasis suppression and hypothesized that critical protein interactions in this region would explain its function. Phosphorylation status at S237 regulates BRMS1 protein interactions related to a variety of biological processes, phenotypes [cell cycle (e.g., CDKN2A), DNA repair (e.g., BRCA1)], and metastasis [(e.g., TCF2 and POLE2)]. Presence of S237 also directly decreased MDA-MB-231 breast carcinoma migration in vitro and metastases in vivo. The results add significantly to our understanding of how BRMS1 interactions with Sin3/HDAC complexes regulate metastasis and expand insights into BRMS1's molecular role, as they demonstrate BRMS1 C-terminus involvement in distinct protein-protein interactions.

Multiple roles for PARP1 in ALC1-dependent nucleosome remodeling.

The SNF2 family ATPase Amplified in Liver Cancer 1 (ALC1) is the only chromatin remodeling enzyme with a poly(ADP-ribose) (PAR) binding macrodomain. ALC1 functions together with poly(ADP-ribose) polymerase PARP1 to remodel nucleosomes. Activation of ALC1 cryptic ATPase activity and the subsequent nucleosome remodeling requires binding of its macrodomain to PAR chains synthesized by PARP1 and NAD+ A key question is whether PARP1 has a role(s) in ALC1-dependent nucleosome remodeling beyond simply synthesizing the PAR chains needed to activate the ALC1 ATPase. Here, we identify PARP1 separation-of-function mutants that activate ALC1 ATPase but do not support nucleosome remodeling by ALC1. Investigation of these mutants has revealed multiple functions for PARP1 in ALC1-dependent nucleosome remodeling and provides insights into its multifaceted role in chromatin remodeling.

The disordered regions of the methyltransferase SETD2 govern its function by regulating its proteolysis and phase separation.

SETD2 is an important methyltransferase that methylates crucial substrates such as histone H3, tubulin, and STAT1 and also physically interacts with transcription and splicing regulators such as Pol II and various hnRNPs. Of note, SETD2 has a functionally uncharacterized extended N-terminal region, the removal of which leads to its stabilization. How this region regulates SETD2 half-life is unclear. Here we show that SETD2 consists of multiple long disordered regions across its length that cumulatively destabilize the protein by facilitating its proteasomal degradation. SETD2 disordered regions can reduce the half-life of the yeast homolog Set2 in mammalian cells as well as in yeast, demonstrating the importance of intrinsic structural features in regulating protein half-life. In addition to the shortened half-life, by performing fluorescence recovery after photobleaching assay we found that SETD2 forms liquid droplets in vivo, another property associated with proteins that contain disordered regions. The phase-separation behavior of SETD2 is exacerbated upon the removal of its N-terminal segment and results in activator-independent histone H3K36 methylation. Our findings reveal that disordered region-facilitated proteolysis is an important mechanism governing SETD2 function.

K63-linked ubiquitination of DYRK1A by TRAF2 alleviates Sprouty 2-mediated degradation of EGFR.

Dual specificity tyrosine phosphorylation regulated kinase 1A, DYRK1A, functions in multiple cellular pathways, including signaling, endocytosis, synaptic transmission, and transcription. Alterations in dosage of DYRK1A leads to defects in neurogenesis, cell growth, and differentiation, and may increase the risk of certain cancers. DYRK1A localizes to a number of subcellular structures including vesicles where it is known to phosphorylate a number of proteins and regulate vesicle biology. However, the mechanism by which it translocates to vesicles is poorly understood. Here we report the discovery of TRAF2, an E3 ligase, as an interaction partner of DYRK1A. Our data suggest that TRAF2 binds to PVQE motif residing in between the PEST and histidine repeat domain (HRD) of DYRK1A protein, and mediates K63-linked ubiquitination of DYRK1A. This results in translocation of DYRK1A to the vesicle membrane. DYRK1A increases phosphorylation of Sprouty 2 on vesicles, leading to the inhibition of EGFR degradation, and depletion of TRAF2 expression accelerates EGFR degradation. Further, silencing of DYRK1A inhibits the growth of glioma cells mediated by TRAF2. Collectively, these findings suggest that the axis of TRAF2-DYRK1A-Sprouty 2 can be a target for new therapeutic development for EGFR-mediated human pathologies.

The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain.

Heterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2, by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.

Harnessing Ionic Selectivity in Acetyltransferase Chemoproteomic Probes.

Chemical proteomics provides a powerful strategy for the high-throughput assignment of enzyme function or inhibitor selectivity. However, identifying optimized probes for an enzyme family member of interest and differentiating signal from the background remain persistent challenges in the field. To address this obstacle, here we report a physiochemical discernment strategy for optimizing chemical proteomics based on the coenzyme A (CoA) cofactor. First, we synthesize a pair of CoA-based sepharose pulldown resins differentiated by a single negatively charged residue and find this change alters their capture properties in gel-based profiling experiments. Next, we integrate these probes with quantitative proteomics and benchmark analysis of "probe selectivity" versus traditional "competitive chemical proteomics." This reveals that the former is well-suited for the identification of optimized pulldown probes for specific enzyme family members, while the latter may have advantages in discovery applications. Finally, we apply our anionic CoA pulldown probe to evaluate the selectivity of a recently reported small molecule N-terminal acetyltransferase inhibitor. These studies further validate the use of physical discriminant strategies in chemoproteomic hit identification and demonstrate how CoA-based chemoproteomic probes can be used to evaluate the selectivity of small molecule protein acetyltransferase inhibitors, an emerging class of preclinical therapeutic agents.

Identification of stem cells from large cell populations with topological scoring.

Machine learning and topological analysis methods are becoming increasingly used on various large-scale omics datasets. Modern high dimensional flow cytometry data sets share many features with other omics datasets like genomics and proteomics. For example, genomics or proteomics datasets can be sparse and have high dimensionality, and flow cytometry datasets can also share these features. This makes flow cytometry data potentially a suitable candidate for employing machine learning and topological scoring strategies, for example, to gain novel insights into patterns within the data. We have previously developed a Topological Score (TopS) and implemented it for the analysis of quantitative protein interaction network datasets. Here we show that TopS approach for large scale data analysis is applicable to the analysis of a previously described flow cytometry sorted human hematopoietic stem cell dataset. We demonstrate that TopS is capable of effectively sorting this dataset into cell populations and identify rare cell populations. We demonstrate the utility of TopS when coupled with multiple approaches including topological data analysis, X-shift clustering, and t-Distributed Stochastic Neighbor Embedding (t-SNE). Our results suggest that TopS could be effectively used to analyze large scale flow cytometry datasets to find rare cell populations.

Driving integrative structural modeling with serial capture affinity purification.

Streamlined characterization of protein complexes remains a challenge for the study of protein interaction networks. Here we describe serial capture affinity purification (SCAP), in which two separate proteins are tagged with either the HaloTag or the SNAP-tag, permitting a multistep affinity enrichment of specific protein complexes. The multifunctional capabilities of this protein-tagging system also permit in vivo validation of interactions using acceptor photobleaching Förster resonance energy transfer and fluorescence cross-correlation spectroscopy quantitative imaging. By coupling SCAP to cross-linking mass spectrometry, an integrative structural model of the complex of interest can be generated. We demonstrate this approach using the Spindlin1 and SPINDOC protein complex, culminating in a structural model with two SPINDOC molecules docked on one SPIN1 molecule. In this model, SPINDOC interacts with the SPIN1 interface previously shown to bind a lysine and arginine methylated sequence of histone H3. Our approach combines serial affinity purification, live cell imaging, and cross-linking mass spectrometry to build integrative structural models of protein complexes.