Repeat modules and N-linked glycans define structure and antigenicity of a critical enterotoxigenic E. coli adhesin.

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of cases of infectious diarrhea annually, predominantly in children from low-middle income regions. Notably, in children, as well as human volunteers challenged with ETEC, diarrheal severity is significantly increased severity in blood group A (bgA) individuals. EtpA, is a secreted glycoprotein adhesin that functions as a blood group A lectin to promote critical interactions between ETEC and blood group A glycans on intestinal epithelia for effective bacterial adhesion and toxin delivery. EtpA is highly immunogenic resulting in robust antibody responses following natural infection and experimental challenge of human volunteers with ETEC. To understand how EtpA directs ETEC-blood group A interactions and stimulates adaptive immunity, we mutated EtpA, mapped its glycosylation by mass-spectrometry (MS), isolated polyclonal (pAbs) and monoclonal antibodies (mAbs) from vaccinated mice and ETEC-infected human volunteers, and determined structures of antibody-EtpA complexes by cryo-electron microscopy. Both bgA and mAbs that inhibited EtpA-bgA interactions and ETEC adhesion, bound to the C-terminal repeat domain highlighting this region as crucial for ETEC pathogen-host interaction. MS analysis uncovered extensive and heterogeneous N-linked glycosylation of EtpA and cryo-EM structures revealed that mAbs directly engage these unique glycan containing epitopes. Finally, electron microscopy-based polyclonal epitope mapping revealed antibodies targeting numerous distinct epitopes on N and C-terminal domains, suggesting that EtpA vaccination generates responses against neutralizing and decoy regions of the molecule. Collectively, we anticipate that these data will inform our general understanding of pathogen-host glycan interactions and adaptive immunity relevant to rational vaccine subunit design.

Analysis of the Tropism of SARS-CoV-2 Based on the Host Interactome of the Spike Protein.

The ß-coronavirus SARS-CoV-2 causes severe acute respiratory syndrome (COVID-19) in humans. It enters and infects epithelial airway cells upon binding of the receptor binding domain (RBD) of the virus entry protein spike to the host receptor protein Angiotensin Converting Enzyme 2 (ACE2). Here, we used coimmunoprecipitation coupled with bottom-up mass spectrometry to identify host proteins that engaged with the spike protein in human bronchial epithelial cells (16HBEo-). We found that the spike protein bound to extracellular laminin and thrombospondin and endoplasmatic reticulum (ER)-resident DJB11 and FBX2 proteins. The ER-resident proteins UGGT1, CALX, HSP7A, and GRP78/BiP bound preferentially to the original Wuhan D614 over the mutated G614 spike protein in the more rapidly spreading Alpha SARS-CoV-2 strain. The increase in protein binding to the D614 spike might be explained by higher accessibility of cryptic sites in "RDB open" and "S2 only" D614 spike protein conformations and may enable SARS-CoV-2 to infect additional, ACE2-negative cell types. Moreover, a novel proteome-based cell type set enrichment analysis (pCtSEA) found that host factors like laminin might render additional cell types such as macrophages and epithelial cells in the nephron permissive to SARS-CoV-2 infection.

Comparative membrane proteomics reveals diverse cell regulators concentrated at the nuclear envelope.

The nuclear envelope (NE) is a subdomain of the ER with prominent roles in nuclear organization, which are largely mediated by its distinctive protein composition. We developed methods to reveal low-abundance transmembrane (TM) proteins concentrated at the NE relative to the peripheral ER. Using label-free proteomics that compared isolated NEs with cytoplasmic membranes, we first identified proteins with apparent NE enrichment. In subsequent authentication, ectopically expressed candidates were analyzed by immunofluorescence microscopy to quantify their targeting to the NE in cultured cells. Ten proteins from a validation set were found to associate preferentially with the NE, including oxidoreductases, enzymes for lipid biosynthesis, and regulators of cell growth and survival. We determined that one of the validated candidates, the palmitoyltransferase Zdhhc6, modifies the NE oxidoreductase Tmx4 and thereby modulates its NE levels. This provides a functional rationale for the NE concentration of Zdhhc6. Overall, our methodology has revealed a group of previously unrecognized proteins concentrated at the NE and additional candidates. Future analysis of these can potentially unveil new mechanistic pathways associated with the NE.

DeGlyPHER: Highly sensitive site-specific analysis of N-linked glycans on proteins.

Traditional mass spectrometry-based glycoproteomic approaches have been widely used for site-specific N-glycoform analysis, but a large amount of starting material is needed to obtain sampling that is representative of the vast diversity of N-glycans on glycoproteins. These methods also often include a complicated workflow and very challenging data analysis. These limitations have prevented glycoproteomics from being adapted to high-throughput platforms, and the sensitivity of the analysis is currently inadequate for elucidating N-glycan heterogeneity in clinical samples. Heavily glycosylated spike proteins of enveloped viruses, recombinantly expressed as potential vaccines, are prime targets for glycoproteomic analysis. Since the immunogenicity of spike proteins may be impacted by their glycosylation patterns, site-specific analysis of N-glycoforms provides critical information for vaccine design. Using recombinantly expressed soluble HIV Env trimer, we describe DeGlyPHER, a modification of our previously reported sequential deglycosylation strategy to yield a "single-pot" process. DeGlyPHER is an ultrasensitive, simple, rapid, robust, and efficient approach for site-specific analysis of protein N-glycoforms, that we developed for analysis of limited quantities of glycoproteins.

Comparative membrane proteomics reveals diverse cell regulators concentrated at the nuclear envelope.

The nuclear envelope (NE) is a subdomain of the ER with prominent roles in nuclear organization, largely mediated by its distinctive protein composition. We developed methods to reveal novel, low abundance transmembrane (TM) proteins concentrated at the NE relative to the peripheral ER. Using label-free proteomics that compared isolated NEs to cytoplasmic membranes, we first identified proteins with apparent NE enrichment. In subsequent authentication, ectopically expressed candidates were analyzed by immunofluorescence microscopy to quantify their targeting to the NE in cultured cells. Ten proteins from a validation set were found to associate preferentially with the NE, including oxidoreductases, enzymes for lipid biosynthesis and regulators of cell growth and survival. We determined that one of the validated candidates, the palmitoyltransferase Zdhhc6, modifies the NE oxidoreductase Tmx4 and thereby modulates its NE levels. This provides a functional rationale for the NE concentration of Zdhhc6. Overall, our methodology has revealed a group of previously unrecognized proteins concentrated at the NE and additional candidates. Future analysis of these can potentially unveil new mechanistic pathways associated with the NE.

Shared and Distinctive Neighborhoods of Emerin and Lamin B Receptor Revealed by Proximity Labeling and Quantitative Proteomics.

Emerin and lamin B receptor (LBR) are abundant transmembrane proteins of the nuclear envelope that are concentrated at the inner nuclear membrane (INM). Although both proteins interact with chromatin and nuclear lamins, they have distinctive biochemical and functional properties. Here, we have deployed proximity labeling using the engineered biotin ligase TurboID (TbID) and quantitative proteomics to compare the neighborhoods of emerin and LBR in cultured mouse embryonic fibroblasts. Our analysis revealed 232 high confidence proximity partners that interact selectively with emerin and/or LBR, 49 of which are shared by both. These included previously characterized NE-concentrated proteins, as well as a host of additional proteins not previously linked to emerin or LBR functions. Many of these are TM proteins of the ER, including two E3 ubiquitin ligases. Supporting these results, we found that 11/12 representative proximity relationships identified by TbID also were detected at the NE with the proximity ligation assay. Overall, this work presents methodology that may be used for large-scale mapping of the landscape of the INM and reveals a group of new proteins with potential functional connections to emerin and LBR.

Interactome analysis illustrates diverse gene regulatory processes associated with LIN28A in human iPS cell-derived neural progenitor cells.

A single protein can be multifaceted depending on the cellular contexts and interacting molecules. LIN28A is an RNA-binding protein that governs developmental timing, cellular proliferation, differentiation, stem cell pluripotency, and metabolism. In addition to its best-known roles in microRNA biogenesis, diverse molecular roles have been recognized. In the nervous system, LIN28A is known to play critical roles in proliferation and differentiation of neural progenitor cells (NPCs). We profiled the endogenous LIN28A-interacting proteins in NPCs differentiated from human induced pluripotent stem (iPS) cells using immunoprecipitation and liquid chromatography-tandem mass spectrometry. We identified over 500 LIN28A-interacting proteins, including 156 RNA-independent interactors. Functions of these proteins span a wide range of gene regulatory processes. Prompted by the interactome data, we revealed that LIN28A may impact the subcellular distribution of its interactors and stress granule formation upon oxidative stress. Overall, our analysis opens multiple avenues for elaborating molecular roles and characteristics of LIN28A.

DeGlyPHER: An Ultrasensitive Method for the Analysis of Viral Spike N-Glycoforms.

Viruses can evade the host immune system by displaying numerous glycans on their surface "spike-proteins" that cover immune epitopes. We have developed an ultrasensitive "single-pot" method to assess glycan occupancy and the extent of glycan processing from high-mannose to complex forms at each N-glycosylation site. Though aimed at characterizing glycosylation of viral spike-proteins as potential vaccines, this method is applicable for the analysis of site-specific glycosylation of any glycoprotein.

Protein Footprinting via Covalent Protein Painting Reveals Structural Changes of the Proteome in Alzheimer's Disease.

Misfolding and aggregation of amyloid-ß peptide and hyperphosphorylated tau are molecular markers of Alzheimer's disease (AD), and although the 3D structures of these aberrantly folded proteins have been visualized in exquisite detail, no method has been able to survey protein folding across the proteome in AD. Here, we present covalent protein painting (CPP), a mass spectrometry-based protein footprinting approach to quantify the accessibility of lysine ε-amines for covalent modification at the surface of natively folded proteins. We used CPP to survey the reactivity of 2645 lysine residues and therewith the structural proteome of HEK293T cells and found that reactivity increased upon mild heat shock. CPP revealed that the accessibility of lysine residues for covalent modification in tubulin-ß (TUBB), in succinate dehydrogenase (SHDB), and in amyloid-ß peptide (Aß) is altered in human postmortem brain samples of patients with neurodegenerative diseases. The structural alterations of TUBB and SHDB in patients with AD, dementia with Lewy bodies (DLB), or both point to broader perturbations of the 3D proteome beyond Aß and hyperphosphorylated tau.

Altered network and rescue of human neurons derived from individuals with early-onset genetic epilepsy.

Early-onset epileptic encephalopathies are severe disorders often associated with specific genetic mutations. In this context, the CDKL5 deficiency disorder (CDD) is a neurodevelopmental condition characterized by early-onset seizures, intellectual delay, and motor dysfunction. Although crucial for proper brain development, the precise targets of CDKL5 and its relation to patients' symptoms are still unknown. Here, induced pluripotent stem cells derived from individuals deficient in CDKL5 protein were used to generate neural cells. Proteomic and phosphoproteomic approaches revealed disruption of several pathways, including microtubule-based processes and cytoskeleton organization. While CDD-derived neural progenitor cells have proliferation defects, neurons showed morphological alterations and compromised glutamatergic synaptogenesis. Moreover, the electrical activity of CDD cortical neurons revealed hyperexcitability during development, leading to an overly synchronized network. Many parameters of this hyperactive network were rescued by lead compounds selected from a human high-throughput drug screening platform. Our results enlighten cellular, molecular, and neural network mechanisms of genetic epilepsy that could ultimately promote novel therapeutic opportunities for patients.

The Host Interactome of Spike Expands the Tropism of SARS-CoV-2.

The SARS-CoV-2 virus causes severe acute respiratory syndrome (COVID-19) and has rapidly created a global pandemic. Patients that survive may face a slow recovery with long lasting side effects that can afflict different organs. SARS-CoV-2 primarily infects epithelial airway cells that express the host entry receptor Angiotensin Converting Enzyme 2 (ACE2) which binds to spike protein trimers on the surface of SARS-CoV-2 virions. However, SARS-CoV-2 can spread to other tissues even though they are negative for ACE2. To gain insight into the molecular constituents that might influence SARS-CoV-2 tropism, we determined which additional host factors engage with the viral spike protein in disease-relevant human bronchial epithelial cells (16HBEo-). We found that spike recruited the extracellular proteins laminin and thrombospondin and was retained in the endoplasmatic reticulum (ER) by the proteins DJB11 and FBX2 which support re-folding or degradation of nascent proteins in the ER. Because emerging mutations of the spike protein potentially impact the virus tropism, we compared the interactome of D614 spike with that of the rapidly spreading G614 mutated spike. More D614 than G614 spike associated with the proteins UGGT1, calnexin, HSP7A and GRP78/BiP which ensure glycosylation and folding of proteins in the ER. In contrast to G614 spike, D614 spike was endoproteolytically cleaved, and the N-terminal S1 domain was degraded in the ER even though C-terminal 'S2 only' proteoforms remained present. D614 spike also bound more laminin than G614 spike, which suggested that extracellular laminins may function as co-factor for an alternative, 'S2 only' dependent virus entry. Because the host interactome determines whether an infection is productive, we developed a novel proteome-based cell type set enrichment analysis (pCtSEA). With pCtSEA we determined that the host interactome of the spike protein may extend the tropism of SARS-CoV-2 beyond mucous epithelia to several different cell types, including macrophages and epithelial cells in the nephron. An 'S2 only' dependent, alternative infection of additional cell types with SARS-CoV-2 may impact vaccination strategies and may provide a molecular explanation for a severe or prolonged progression of disease in select COVID-19 patients.

Temporal Quantitative Profiling of Newly Synthesized Proteins during Aß Accumulation.

Accumulation of aggregated amyloid beta (Aß) in the brain is believed to impair multiple cellular pathways and play a central role in Alzheimer's disease pathology. However, how this process is regulated remains unclear. In theory, measuring protein synthesis is the most direct way to evaluate a cell's response to stimuli, but to date, there have been few reliable methods to do this. To identify the protein regulatory network during the development of Aß deposition in AD, we applied a new proteomic technique to quantitate newly synthesized protein (NSP) changes in the cerebral cortex and hippocampus of 2-, 5-, and 9-month-old APP/PS1 AD transgenic mice. This bio-orthogonal noncanonical amino acid tagging analysis combined PALM (pulse azidohomoalanine labeling in mammals) and HILAQ (heavy isotope labeled AHA quantitation) to reveal a comprehensive dataset of NSPs prior to and post Aß deposition, including the identification of proteins not previously associated with AD, and demonstrated that the pattern of differentially expressed NSPs is age-dependent. We also found dysregulated vesicle transportation networks including endosomal subunits, coat protein complex I (COPI), and mitochondrial respiratory chain throughout all time points and two brain regions. These results point to a pathological dysregulation of vesicle transportation which occurs prior to Aß accumulation and the onset of AD symptoms, which may progressively impact the entire protein network and thereby drive neurodegeneration. This study illustrates key pathway regulation responses to the development of AD pathogenesis by directly measuring the changes in protein synthesis and provides unique insights into the mechanisms that underlie AD.

Quantitative analysis of global protein stability rates in tissues.

Protein degradation is an essential mechanism for maintaining proteostasis in response to internal and external perturbations. Disruption of this process is implicated in many human diseases. We present a new technique, QUAD (Quantification of Azidohomoalanine Degradation), to analyze the global degradation rates in tissues using a non-canonical amino acid and mass spectrometry. QUAD analysis reveals that protein stability varied within tissues, but discernible trends in the data suggest that cellular environment is a major factor dictating stability. Within a tissue, different organelles and protein functions were enriched with different stability patterns. QUAD analysis demonstrated that protein stability is enhanced with age in the brain but not in the liver. Overall, QUAD allows the first global quantitation of protein stability rates in tissues, which will allow new insights and hypotheses in basic and translational research.

Impact of the Identification Strategy on the Reproducibility of the DDA and DIA Results.

Data-independent acquisition (DIA) is a promising technique for the proteomic analysis of complex protein samples. A number of studies have claimed that DIA experiments are more reproducible than data-dependent acquisition (DDA), but these claims are unsubstantiated since different data analysis methods are used in the two methods. Data analysis in most DIA workflows depends on spectral library searches, whereas DDA typically employs sequence database searches. In this study, we examined the reproducibility of the DIA and DDA results using both sequence database and spectral library search. The comparison was first performed using a cell lysate and then extended to an interactome study. Protein overlap among the technical replicates in both DDA and DIA experiments was 30% higher with library-based identifications than with sequence database identifications. The reproducibility of quantification was also improved with library search compared to database search, with the mean of the coefficient of variation decreasing more than 30% and a reduction in the number of missing values of more than 35%. Our results show that regardless of the acquisition method, higher identification and quantification reproducibility is observed when library search was used.

Improving Proteomics Data Reproducibility with a Dual-Search Strategy.

Mass spectrometry-based proteomics is an invaluable tool for addressing important biological questions. Data-dependent acquisition methods effectuate stochastic acquisition of data in complex mixtures, which results in missing identifications across replicates. We developed a search approach that improves the reproducibility of data acquired from any mass spectrometer. In our approach, a spectral library is built from the identification results from a database search, and then, the library is used to research the same data files to obtain the final result. We showed that higher identification and quantification reproducibility is achieved with the dual-search approach than with a typical database search. Four datasets with different complexity were compared: (1) data from a cell lysate study performed in our lab, (2) data from an interactome study performed in our lab, (3) a publicly available extracellular vesicles dataset, and (4) a publicly available phosphoproteomics dataset. Our results show that the dual-search approach can be widely and easily used to improve data quality in proteomics data.

Comparison of CRISPR Genomic Tagging for Affinity Purification and Endogenous Immunoprecipitation Coupled with Quantitative Mass Spectrometry To Identify the Dynamic AMPKα2 Interactome.

Recent advances in genome editing technologies have enabled the insertion of epitope tags at endogenous loci with relative efficiency. We describe an approach for investigation of protein interaction dynamics of the AMP-activated kinase complex AMPK using a catalytic subunit AMPKα2 (PRKAA2 gene) as the bait, based on CRISPR/Cas9-mediated genome editing coupled to stable isotope labeling in cell culture, multidimensional protein identification technology, and computational and statistical analyses. Furthermore, we directly compare this genetic epitope tagging approach to endogenous immunoprecipitations of the same gene under homologous conditions to assess differences in observed interactors. Additionally, we directly compared each enrichment strategy in the genetically modified cell-line with two separate endogenous antibodies. For each approach, we analyzed the interaction profiles of this protein complex under basal and activated states, and after implementing the same analytical, computational, and statistical analyses, we found that high-confidence protein interactors vary greatly with each method and between commercially available endogenous antibodies.

Proteomics INTegrator (PINT): An Online Tool To Store, Query, and Visualize Large Proteomics Experiment Results.

The characterization of complex biological systems based on high-throughput protein quantification through mass spectrometry commonly involves differential expression analysis between replicate samples originating from different experimental conditions. Here we present Proteomics INTegrator (PINT), a new user-friendly Web-based platform-independent system to store, visualize, and query proteomics experiment results. PINT provides an extremely flexible query interface that allows advanced Boolean algebra-based data filtering of many different proteomics features such as confidence values, abundance levels or ratios, data set overlaps, sample characteristics, as well as UniProtKB annotations, which are transparently incorporated into the system. In addition, PINT allows developers to incorporate data visualization and analysis tools, such as PSEA-Quant and Reactome pathway analysis, for data set enrichment analysis. PINT serves as a centralized hub for large-scale proteomics data and as a platform for data analysis, facilitating the interpretation of proteomics results and expediting biologically relevant conclusions.

Identification of new transmembrane proteins concentrated at the nuclear envelope using organellar proteomics of mesenchymal cells.

The double membrane nuclear envelope (NE), which is contiguous with the ER, contains nuclear pore complexes (NPCs) - the channels for nucleocytoplasmic transport, and the nuclear lamina (NL) - a scaffold for NE and chromatin organization. Since numerous human diseases linked to NE proteins occur in mesenchyme-derived cells, we used proteomics to characterize NE and other subcellular fractions isolated from mesenchymal stem cells and from adipocytes and myocytes. Based on spectral abundance, we calculated enrichment scores for proteins in the NE fractions. We demonstrated by quantitative immunofluorescence microscopy that five little-characterized proteins with high enrichment scores are substantially concentrated at the NE, with Itprip exposed at the outer nuclear membrane, Smpd4 enriched at the NPC, and Mfsd10, Tmx4, and Arl6ip6 likely residing in the inner nuclear membrane. These proteins provide new focal points for studying the functions of the NE. Moreover, our datasets provide a resource for evaluating additional potential NE proteins.

Understanding molecular mechanisms of disease through spatial proteomics.

Mammalian cells are organized into different compartments that separate and facilitate physiological processes by providing specialized local environments and allowing different, otherwise incompatible biological processes to be carried out simultaneously. Proteins are targeted to these subcellular locations where they fulfill specialized, compartment-specific functions. Spatial proteomics aims to localize and quantify proteins within subcellular structures.

Structural Analysis of Hippocampal Kinase Signal Transduction.

Kinases are a major clinical target for human diseases. Identifying the proteins that interact with kinases in vivo will provide information on unreported substrates and will potentially lead to more specific methods for therapeutic kinase regulation. Here, endogenous immunoprecipitations of evolutionally distinct kinases (i.e., Akt, ERK2, and CAMK2) from rodent hippocampi were analyzed by mass spectrometry to generate three highly confident kinase protein-protein interaction networks. Proteins of similar function were identified in the networks, suggesting a universal model for kinase signaling complexes. Protein interactions were observed between kinases with reported symbiotic relationships. The kinase networks were significantly enriched in genes associated with specific neurodevelopmental disorders providing novel structural connections between these disease-associated genes. To demonstrate a functional relationship between the kinases and the network, pharmacological manipulation of Akt in hippocampal slices was shown to regulate the activity of potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel(HCN1), which was identified in the Akt network. Overall, the kinase protein-protein interaction networks provide molecular insight of the spatial complexity of in vivo kinase signal transduction which is required to achieve the therapeutic potential of kinase manipulation in the brain.