Research Articles
- Research Article
The SARS-CoV2 envelope differs from host cells, exposes procoagulant lipids, and is disrupted in vivo by oral rinses
The lipid envelope of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an essential component of the virus; however, its molecular composition is undetermined. Addressing this knowledge gap could support the design of antiviral agents as well as further our understanding of viral-host protein interactions, infectivity, pathogenicity, and innate immune system clearance. Lipidomics revealed that the virus envelope comprised mainly phospholipids (PLs), with some cholesterol and sphingolipids, and with cholesterol/phospholipid ratio similar to lysosomes. - Research Article
Whole-exome sequencing reveals damaging gene variants associated with hypoalphalipoproteinemia
Low levels of high density lipoprotein-cholesterol (HDL-C) are associated with an elevated risk of arteriosclerotic coronary heart disease. Heritability of HDL-C levels is high. In this research discovery study, we used whole-exome sequencing to identify damaging gene variants that may play significant roles in determining HDL-C levels. We studied 204 individuals with a mean HDL-C level of 27.8 ± 6.4 mg/dl (range: 4–36 mg/dl). Data were analyzed by statistical gene burden testing and by filtering against candidate gene lists. - Research Article
Sulfatide with ceramide composed of phytosphingosine (t18:0) and 2-hydroxy FAs in renal intercalated cells
Diverse molecular species of sulfatide with differences in FA lengths, unsaturation degrees, and hydroxylation statuses are expressed in the kidneys. However, the physiological functions of specific sulfatide species in the kidneys are unclear. Here, we evaluated the distribution of specific sulfatide species in the kidneys and their physiological functions. Electron microscopic analysis of kidneys of Cst-deficient mice lacking sulfatide showed vacuolar accumulation in the cytoplasm of intercalated cells in the collecting duct, whereas the proximal and distal tubules were unchanged. - Research Article
Genetic dissection in mice reveals a dynamic crosstalk between the delivery pathways of vitamin A
Vitamin A is distributed within the body to support chromophore synthesis in the eyes and retinoid signaling in most other tissues. Two pathways exist for the delivery of vitamin A: the extrinsic pathway transports dietary vitamin A in lipoproteins from intestinal enterocytes to tissues, while the intrinsic pathway distributes vitamin A from hepatic stores bound to serum retinol binding protein (RBP). Previously, the intestine-specific homeodomain transcription factor (ISX) and the RBP receptor STRA6 were identified as gatekeepers of these pathways; however, it is not clear how mutations in the corresponding genes affect retinoid homeostasis. - Research Article
Sortilin enhances secretion of apolipoprotein(a) through effects on apolipoprotein B secretion and promotes uptake of lipoprotein(a)
Elevated plasma lipoprotein(a) (Lp(a)) is an independent, causal risk factor for atherosclerotic cardiovascular disease and calcific aortic valve stenosis. Lp(a) is formed in or on hepatocytes from successive noncovalent and covalent interactions between apo(a) and apoB, although the subcellular location of these interactions and the nature of the apoB-containing particle involved remain unclear. Sortilin, encoded by the SORT1 gene, modulates apoB secretion and LDL clearance. We used a HepG2 cell model to study the secretion kinetics of apo(a) and apoB. - Research Article
Proteomic Analysis Uncovers Measles Virus Protein C Interaction With p65–iASPP Protein Complex
In Brief Control of measles virus infection and measles-based oncolytic therapy are possible, thanks to the existence of a safe and efficient live attenuated vaccine. Molecular mechanisms that make this vaccine to be so efficient are yet to be determined. We show that the measles C protein is responsible for the establishment of complex networks of interactions with the host cell. We suggest that the C protein binding to the p65–iASPP protein complex controls the host cell death and innate immunity pathways. - Research Article
Dynamic Changes to the Skeletal Muscle Proteome and Ubiquitinome Induced by the E3 Ligase, ASB2β
In Brief The E3 ubiquitin ligase ASB2β has been identified as a regulator of skeletal muscle mass. To gain insights into potential mechanisms of action, mouse muscles expressing a Flag-tagged ASB2β were investigated using quantitative proteomic methods. The results identified ASB2β-induced changes in the abundance and ubiquitination of proteins associated with mitochondria, the sarcomere, and the cytoskeleton. Additional in vitro studies identified novel putative ASB2β target substrates. The results highlight the complex relationship between protein abundance and ubiquitination in ASB2β-mediated muscle adaptation. - Research Article
New Proteomic Signatures to Distinguish Between Zika and Dengue Infections
In Brief Differentiation between the mosquito-borne Zika and dengue Flavivirus infections is clinically important for correct treatment, but remains challenging. We used mass spectrometry to quantify expression levels for 277 proteins measured in serum samples from 62 patients, providing a resource to the community. We identified 13 proteins with significant differential expression between the closely related types of infections. Most of the proteins link to pregnancy and brain function. We also identified expression signatures that mark ambiguous infections with respect to temporal differences. - Research Article
Protein Phosphorylation in Depolarized Synaptosomes: Dissecting Primary Effects of Calcium from Synaptic Vesicle Cycling
In Brief Analysis of protein phosphorylation in isolated nerve terminals (synaptosomes) treated with C. botulinum neurotoxins (BoNT) to inhibit synaptic vesicle (SV) cycling reveals phosphorylation events that are primarily dependent on depolarization-induced Ca2+ influx and those that also require active SV-cycling machinery. In particular, SV-cycling-dependent phosphorylation sites on synaptobrevin (Vamp2), syntaxin-1 (Stx1a), and cannabinoid receptor-1 (Cnr1) are capable of changing the rate of exo- and endocytosis in cultured hippocampal neurons. - Research Article
Quantitative Proteomics and Phosphoproteomics Support a Role for Mut9-Like Kinases in Multiple Metabolic and Signaling Pathways in Arabidopsis
In Brief The MUT9-like kinases are a family of plant-specific nuclear-localized kinases with roles in diverse signaling pathways, including light sensing, phytohormone perception, and the circadian clock. The proteome and phosphoproteome of compound mlk mutant seedlings have been determined under light and dark conditions. These experiments identify new roles for these kinases regulating secondary plant metabolism and stress responses, tested through metabolite analysis and assaying seedling sensitivity to DNA damaging agents. - Research Article
Proximity-dependent Mapping of the Androgen Receptor Identifies Kruppel-like Factor 4 as a Functional Partner
In Brief A proximity interaction network for the androgen receptor (AR) was obtained from androgen-responsive prostate cancer cells. A total of 267 candidates were identified, most associating following ligand stimulation, including Krüppel-like factor 4 (KLF4). KLF4 and AR were found to colocalize genome-wide on 4097 genes including PSA (KLK3), for which KLF4 acts as a repressor, without regulating the expression of AR. These results are instrumental to further dissect the molecular mechanisms underlying androgen signaling in prostate cells. - Technological Innovation and Resources
Stable Isotope Labeling of Amino Acids in Flies (SILAF) Reveals Differential Phosphorylation of Mitochondrial Proteins Upon Loss of OXPHOS Subunits
In Brief We advanced a fully composed food source for Drosophila melanogaster into a highly efficient, versatile, and cheap labeling method, termed SILAF. The larval proteome incorporates more than 99% heavy lysine-6 label within 6 days, while the adult fly metabolism allows protein turnover studies. We obtained a phosphoproteome with site-specific occupancy in a fly model of mitochondrial metabolic disease, highlighting the regulation of two novel conserved phosphosites on subunits of the electron transport chain. - Research Article
Secretome and Comparative Proteomics of Yersinia pestis Identify Two Novel E3 Ubiquitin Ligases That Contribute to Plague Virulence
In Brief Rapid adaption to the environments is critical for microbes to establish infection. Quantitative proteome and secretome analyses of Y. pestis grown under conditions mimicking its two typical natural niches were performed to understand the adaption strategies of this deadly pathogen. We identified three secreted proteins that can be translocated into host cells and further demonstrated that two of them show strong E3 ubiquitin ligase activity and contribute significantly to the virulence of Y. pestis. - Research Article
Proteomic Profiling of Gastric Signet Ring Cell Carcinoma Tissues Reveals Characteristic Changes of the Complement Cascade Pathway
In Brief This study took advantages of LCM and DIA-MS, generating a data set in the context of different subtypes of gastric tumors, globally and precisely. It was discovered for the first time that the complement cascade in SRCC tumors was specifically activated compared with AC. - Research Article
Quantitative Proteomics Reveals that the OGT Interactome Is Remodeled in Response to Oxidative Stress
In Brief The goal of these studies was to provide insight into the regulation of the O-GlcNAc transferase (OGT) basally and in response to oxidative stress, as well as the role that O-GlcNAc plays in promoting cytoprotection. Using quantitative proteomics, the basal and injury-induced interactome of OGT has been defined and validated. Protein interactors are anticipated to regulate either the activity or substrate targeting of OGT, or to be substrates of OGT, thus affecting cytoprotection. - Research Article
Integrative Proteomic and Metabolomic Analysis Reveals Metabolic Phenotype in Mice With Cardiac-Specific Deletion of Natriuretic Peptide Receptor A
In Brief Metabolomic analysis in mice revealed that natriuretic peptide receptor A (NPRA) is mainly involved in nucleotide biosynthesis and histidine metabolism in cardiac tissues, and in creatine metabolism, TCA cycle and pentose phosphate pathway in the plasma. Furthermore, proteomics revealed that Cox7c, Cox7b, ATP5J2, Uqcr10, and Myh7 play a vital role in the regulation of metabolic pathway. Together, deterioration of NPRA results in metabolic dysfunction involved with a protein and metabolite-interacting pathway. - Research Article
Integrated Redox Proteomic Analysis Highlights New Mechanisms of Sensitivity to Silver Nanoparticles
In Brief Silver nanoparticles (AgNPs) are widely used nanomaterials, but the molecular mechanisms underlying their activity remain elusive. Here, we show using time course studies that AgNP-sensitive lung cells experience broader and more pronounced changes in protein abundance and protein oxidation impacting protein translation and modification, lipid metabolism, bioenergetics, and mitochondrial dynamics. The findings are further validated by imaging of mitochondria using confocal microscopy and transmission electron microscopy. - Research Article
Domain Mapping of Chondroitin/Dermatan Sulfate Glycosaminoglycans Enables Structural Characterization of Proteoglycans
In Brief Glycosaminoglycans (GAGs) remain one of the most challenging posttranslational modifications to study, much due to their structural complexity and heterogeneity, and new methods for analysis are therefore required. We have developed a protocol for enrichment and structural characterization of GAGs of proteoglycans using nLC-MS/MS. We provide detailed information on the nonreducing end, internal, and linkage region GAG domains and use the data to determine an overall GAG structure of chromogranin-A of rat INS-1832/13 cells. - Research Article
Mapping Isoform Abundance and Interactome of the Endogenous TMPRSS2-ERG Fusion Protein by Orthogonal Immunoprecipitation–Mass Spectrometry Assays
In Brief Orthogonal immunoprecipitation-mass spectrometry assays quantified TMPRSS2-ERG fusion protein (∼27,000 copies/cell) and its four distinct isoforms, and revealed that T1E4-ERG isoform accounted for 52 ± 3% of the total ERG in VCaP cells and 50 ± 11% in FFPE prostate cancer tissues. Methionine-truncated and N-acetylated peptide TASSSSDYGQTSK unique for T1/E4 TMPRSS2-ERG fusion was identified. Unlike the N-terminal antibodies, C-terminal antibodies identified 29 ERG-interacting proteins, including mutually exclusive BRG1- and BRM-associated canonical SWI/SNF chromatin remodeling complexes. Clinical perspectives of assays were discussed. - Research Article
In-depth Site-specific Analysis of N-glycoproteome in Human Cerebrospinal Fluid and Glycosylation Landscape Changes in Alzheimer's Disease
In Brief An exploratory glycosylation-based biomarker study has been conducted for in-depth mapping of an overall glycosylation landscape and site-specific alteration in glycoproteome collected from cerebrospinal fluids (CSF) in healthy control and Alzheimer’s disease (AD) subjects. The comparison will shed light on the glycoproteome profile, dominant glycosylation differences and similarities, and some of the interesting glycoprotein candidates with specific glycosylation pattern alterations in AD. - Research Article
Feature Selection Methods for Protein Biomarker Discovery from Proteomics or Multiomics Data
In Brief Untargeted mass spectrometry–based proteomics provides a powerful platform for protein biomarker discovery, but clinical translation depends on the selection of a small number of proteins for verification and validation. We present feature selection methods for protein biomarker selection from proteomics or multiomics data. The algorithms show good performance, enable functional interpretation of the identified markers, and provide alternative choices for each identified marker to facilitate a robust transition to the verification and validation platforms. - Technological Innovation and Resources
Enabling Photoactivated Cross-Linking Mass Spectrometric Analysis of Protein Complexes by Novel MS-Cleavable Cross-Linkers
In Brief Although photochemistry complements residue-specific chemistry through labeling amino acids nonspecifically, existing photo-cross-linking reagents are thus far inapplicable to multisubunit protein complexes owing to low yields and high complexities of photo-cross-linked products. The development of the three sulfoxide-containing MS-cleavable photoreactive SDASO cross-linkers permits MSn-based analytical workflow for accurate identification of photo-cross-linked peptides, enabling complex PPI profiling for the first time. This work has established a solid foundation for future applications of photo-cross-linking in complex XL-MS studies. - Research Article
Calculating Sample Size Requirements for Temporal Dynamics in Single-Cell Proteomics
In Brief Cellular development and disease progression are gradual transitions between phenotypic stages. Time-course measurements that explicitly measure this transition are important to discover proteome dynamics. Single-cell measurements are a powerful tool for understanding heterogeneity, especially during phenotypic transitions. Single-cell proteomics measurements are emerging as an available tool to characterize the cellular state. We created a statistical method that predicts the success of an experimental design for temporal dynamics. - Research Article
TMEM67, TMEM237, and Embigin in Complex With Monocarboxylate Transporter MCT1 Are Unique Components of the Photoreceptor Outer Segment Plasma Membrane
In Brief The plasma membrane which envelopes the light-sensitive outer segment organelle of vertebrate photoreceptor cells plays diverse roles in supporting photoreceptor function and health. Protein correlation profiling of this membrane revealed a surprisingly small number of unique protein components. Among them are TMEM67 and TMEM237, whose mutations are associated with various syndromic ciliopathies, and embigin found to be associated with the monocarboxylate transporter MCT1. The MCT1–embigin complex likely facilitates lactate transport through this cellular compartment. - Research Article
HLA Class II Presentation Is Specifically Altered at Elevated Temperatures in the B-Lymphoblastic Cell Line JY
In Brief Here the cellular response in a “fever-mimicking state” was investigated by sampling in parallel the proteome and the HLA class I and II ligandomes. Using quantitative proteomics and immunopeptidomics, we found that a proteomic “fever response” is initiated in B-cells after growing the cells for only 3 days at 40 °C and that it is largely mediated by adaptation in the HLA class II rather than HLA class I system. - Research Article
Structural Investigations of Human A2M Identify a Hollow Native Conformation That Underlies Its Distinctive Protease-Trapping Mechanism
In Brief The native conformation of the protease inhibitor A2M was investigated using negative stain electron microscopy, small-angle X-ray scattering, and cross-linking mass spectrometry. The low-resolution model built from these data describes a hollow tubular configuration that explains several aspects of A2M’s unique trapping mechanism. This model was further validated by two recombinantly expressed A2M mutants, which probed the location of the bait region and demonstrated the existence of a critical interface between A2M’s disulfide-bridged dimers. - Research Article
The Expression of NTAL and Its Protein Interactors Is Associated With Clinical Outcomes in Acute Myeloid Leukemia
In Brief Here, we demonstrated that the knockdown of non–T cell activation linker (NTAL) in acute myeloid leukemia (AML) cells was linked to reduced cell proliferation and survival in vitro and in vivo. In addition, we identified NTAL interactors in AML using label-free protein quantification. NTAL interactors presented a high expression in patients with AML, being associated with a leukemic granulocyte–macrophage progenitor-like state. Finally, NTAL interactors were capable to predict survival in a large subset of patients with AML. These data provide evidence that NTAL and its interactors could represent potential therapeutic targets for granulocyte–macrophage progenitor-like leukemias. - Research Article
The C-Mannosylome of Human Induced Pluripotent Stem Cells Implies a Role for ADAMTS16 C-Mannosylation in Eye Development
In Brief We identified ADAMTS16 as a target protein for C-mannosylation and showed that this modification is needed for proper secretion of ADAMTS16. Targeting a distinct C-mannosyltransferase by CRISPR–Cas9 in medaka fish embryos caused defects in eye development. We conclude that these developmental defects are caused by reduced secretion of ADAMTS16 when C-mannosylation is missing. - Technological Innovation and Resources
GAGrank: Software for Glycosaminoglycan Sequence Ranking Using a Bipartite Graph Model
In Brief We demonstrate GAGrank, an algorithm that uses a bipartite graph model for sequencing glycosaminoglycans from EDD or NETD tandem mass spectra. The process involves first assigning glycosaminoglycan product ions using the GAGfinder algorithm. The second step is to rank possible sequences using GAGrank. We show GAGrank’s ability to sequence isomeric mixtures. - Research Article
Reverse Phase Protein Array Reveals Correlation of Retinoic Acid Metabolism With Cardiomyopathy in Friedreich's Ataxia
In Brief We conducted a sensitive and high-throughput reverse phase protein array to attain protein expression profiles of primary fibroblasts from patients with Friedreich's ataxia (FRDA) and unaffected controls using a pool of 217 validated antibodies. Our extensive bioinformatics analyses correlated differentially expressed (DE) proteins with critical disease parameters. Expression levels of several integrin proteins specifically associated with hearing loss in FRDA. Also, reverse phase protein array data integrated with transcriptome data uncovered defects in retinoic acid metabolism in FRDA samples. - Research Article
Adipose Triglyceride Lipase Loss Promotes a Metabolic Switch in A549 Non–Small Cell Lung Cancer Cell Spheroids
In Brief We here demonstrate that loss of adipose triglyceride lipase (ATGL) in an in-vitro model of 3D lung carcinoma cell (A549) culture leads to a metabolic switch supporting larger spheroid growth and better adaptation to hypoxia. Cancer spheroids lacking ATGL show a higher abundance of glucose transporters, increased glucose uptake and lactate production, and use alternative lipid metabolic pathways. Overall, our data suggest consideration of ATGL as potentially relevant target in lung cancer. - Research Article
Increased Wheat Protein Content via Introgression of an HMW Glutenin Selectively Reshapes the Grain Proteome
In Brief The differences between the grain proteome of three wheat cultivars and corresponding Ay HMW-GS–introgressed near-isogenic lines have been determined. In addition to increased abundance of 1Ay HMW-GS, 115 differentially expressed proteins were also discovered. This revealed that introgression of the 1Ay21∗ HMW-GS increases wheat grain protein content and improves bread-making quality in association with a wider reshaping of the grain proteome network. - Research Article
Revealing the Dynamic Allosteric Changes Required for Formation of the Cysteine Synthase Complex by Hydrogen-Deuterium Exchange MS
In Brief We have used hydrogen/deuterium exchange MS to unveil the allosteric changes occurring during complex formation of CysE and CysK, the last two enzymes of cysteine biosynthetic pathway in bacteria. Significant changes in conformation and dynamics occur in each protein upon complex formation, including long distance intraprotein and interprotein communication. Being absent in mammals, both CysE and CysK represent potential targets for new antibacterial drugs, and our results on the formation and allostery of the complex could help guide drug development. - Research Article
Proteogenomic Assessment of Intraspecific Venom Variability: Molecular Adaptations in the Venom Arsenal of Conus purpurascens
In Brief Cone snail venom is an extensive source of active molecules that have potential pharmacological and biotechnological applications. We employed a top-down functional proteogenomic approach to assess the injected venom of Conus purpurascens. The two distinct venom profiles found will interact differently to target neural pathways aimed to immobilize prey. These venom expression patterns will aid target prediction and the development of conotoxins into drug leads or neural probes. - Research Article
Normothermic Ex-vivo Kidney Perfusion in a Porcine Auto-Transplantation Model Preserves the Expression of Key Mitochondrial Proteins: An Unbiased Proteomics Analysis
In Brief The molecular changes associated with normothermic ex-vivo kidney perfusion (NEVKP) compared with static cold storage were studied using discovery proteomics in a porcine model. NEVKP resulted in increased expression of mitochondrial proteins (ETFB, CPT2) responsible for critical metabolic steps of ATP-synthesis. PPARGC1A, PPARA/D, and RXRA were computationally predicted as upstream regulators of proteins increased in NEVKP and showed increased mRNA expression in NEVKP-treated kidneys. PPAR-family members and their target proteins may represent new therapeutic targets to ameliorate ischemia-reperfusion injury. - Research Article
Nuclear Phosphatidylinositol 3,4,5-Trisphosphate Interactome Uncovers an Enrichment in Nucleolar Proteins
In Brief The polyphosphoinositide (PPIn) phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P3) localizes to the nucleus and nucleolus. Using an affinity enrichment MS approach, the nuclear PtdIns(3,4,5)P3 interactome identified new interaction partners associated with the nucleolus. Among these, the DNA repair PARP1 protein, colocalized to the nucleolus with PtdIns(3,4,5)P3 and showed direct interaction to PPIn via three polybasic regions. The nuclear PtdIns(3,4,5)P3 interactome reported here will serve as a resource to further investigate the molecular mechanisms underlying PtdIns(3,4,5)P3-mediated interactions in the nucleus and nucleolus. - Research Article
Deeply Mining a Universe of Peptides Encoded by Long Noncoding RNAs
In Brief This study proposed a new and effective strategy for the improved discovery and identification of novel SEPs, including the construction of databases maximally collecting all putative small ORFs from human and mouse lncRNA transcripts in NONCODE and the effective enrichment of polypeptides based on 30-kDa molecular weight cutoff (MWCO) membrane and C8 solid-phase extraction column. This effort led to the discovery of 762 novel lncRNA-encoded SEPs from multiple cell lines and tissues. - Research Article
Proteome Profiling of Recombinant DNase Therapy in Reducing NETs and Aiding Recovery in COVID-19 Patients
In Brief Neutrophils contribute to the extracellular DNA pool by forming neutrophil extracellular traps (NETs), which cause sputum thickening, pulmonary inflammation, and hindrance to gaseous exchange during infections. Here, we demonstrate the presence of NETs in sputum from severe COVID-19 patients using mass spectrometry and immunofluorescence analyses. Treatment with clinically approved recombinant human DNase reduced NETs and was associated with improved recovery and reduced inflammation. Targeting NETs using DNase may have significant therapeutic implications in COVID-19 disease and warrants further studies. - Research Article
Age-Associated Proteomic Signatures and Potential Clinically Actionable Targets of Colorectal Cancer
In Brief The incidence of early-onset colorectal cancer (CRC) has been increasing since 1990s, whereas the overall CRC frequency is declining. The underlying mechanisms of age-related clinical differences remain unknown. Here, we reported the proteomic signatures of CRC across age groups. Lots of proteins with adjusted intensities significantly correlated with age. Some proteins were verified as potential clinically actionable targets. This study identifies age-associated proteomic signatures and potential therapeutic targets of CRC and helps to make a precise decision on CRC treatment. - Research Article
Proteome Characterization of Glaucoma Aqueous Humor
In Brief Liu et al. characterized the proteome of aqueous humor from three types of glaucoma. Fifty-seven primary acute angle-closure glaucoma (PAACG), 50 primary chronic angle-closure glaucoma (PCACG), 35 neovascular glaucoma (NVG), and 33 cataract patient samples were analyzed using data-independent analysis and parallel reaction monitoring. Lipid metabolism, immune response, and cell death pathways showed different degrees of activation among the three types of glaucoma but were all higher relative to cataract. SERPIND1 was discovered as a vital protein in glaucoma. - Research Article
A Reductionist Approach Using Primary and Metastatic Cell–Derived Extracellular Vesicles Reveals Hub Proteins Associated with Oral Cancer Prognosis
In Brief A multi-omics strategy was used to map the proteome, miRNA, metabolome, and lipidome of EVs derived from human primary tumor (SCC-9) cells and matched lymph node metastatic (LN1) cells. Differentially abundant molecules associated with the metastatic phenotype were enriched for key processes and pathways. An integrative analysis revealed 11 ‘hub proteins’ that are correlated with reduced survival and tumor aggressiveness in patients with cancer according to public databases. These EV molecules are candidates as prognostic markers in oral cancer. - Research Article
Comparative Host Interactomes of the SARS-CoV-2 Nonstructural Protein 3 and Human Coronavirus Homologs
In Brief SARS-CoV-2 nonstructural protein 3 (nsp3) facilitates virion biogenesis and modulates host ubiquitinylation/ISGylation. The SARS-CoV-2 nsp3 host interactome has not been fully characterized. Using affinity purification–mass spectrometry, we identify interactors of SARS-CoV-2 nsp3 and homologs from four CoV strains. We show the N-terminus of SARS-CoV-2 nsp3 interacts with the transcription factor ATF6 and suppresses its stress response. This work examines the interface between a key CoV protein and host cells, highlighting potential dependencies for antiviral therapeutics. - Research Article
Proteomic Landscape of Exosomes Reveals the Functional Contributions of CD151 in Triple-Negative Breast Cancer
In Brief Using a quantitative proteomics approach, Li et al. characterized the proteomes of triple-negative breast cancer (TNBC) patient–derived serum exosomes and found the tetraspanin CD151 to be significantly enriched. Proteomic analysis of CD151-deleted exosomes and cells showed regulation of ribosomal and complement protein secretion. CD151-deleted exosomes were shown to significantly decrease the migration and invasion of TNBC cells, indicating that exosomal CD151 may be a potential therapeutic target for TNBC. - Research Article
Unbiased Proteomic and Phosphoproteomic Analysis Identifies Response Signatures and Novel Susceptibilities After Combined MEK and mTOR Inhibition in BRAFV600E Mutant Glioma
In Brief BRAFV600E is a key oncogenic driver in glioma, melanoma, and colon cancer. These tumors escape mitogen-activated protein kinase pathway inhibition by upregulating mammalian target of rapamycin signaling. Using comprehensive unbiased proteomic and phosphoproteomic analysis of an in vivo BRAFV600E mutant glioma model treated with inhibitors of both these key pathways, we characterize the tumor and stromal response and suggest additional therapeutic targets for BRAF-driven cancers, including epidermal growth factor receptor and class 1 histone deacetylases. - Research Article
Affinity Proteomics and Deglycoproteomics Uncover Novel EDEM2 Endogenous Substrates and an Integrative ERAD Network
In Brief Various pathologies including neurodegenerative diseases and cancers result from disruptions to or stress of ER homeostasis. One key protein proposed to act in quality control processes maintaining ER homeostasis is EDEM2. Using affinity proteomics and sucrose-density sedimentation, we identified several new EDEM2 partners involved in quality control. Moreover, we defined novel endogenous candidates for EDEM2-dependent degradation by combining glycoproteomics and SILAC-based proteomics. Our data suggest that EDEM2 is involved in ER homeostasis to a greater extent than previously thought. - Research Article
Proteomics, Lipidomics, Metabolomics, and 16S DNA Sequencing of Dental Plaque From Patients With Diabetes and Periodontal Disease
In Brief The human oral cavity houses a complex ecosystem of microbes, some of which have pathogenic influence on the host. Multi-omics analysis of oral plaques revealed key players in microbial communities derived from diabetic and periodontal disease patients. With cross-omic correlation analysis, we found host-specific proteins and associated lipids that were elevated in plaques from periodontal disease patients. Furthermore, this multi-omic approach leads to the finding that oral community member Lautropia mirabilis synthesizes monomethyl phosphatidylethanolamine, an uncommon lipid in oral microbiota. - Research Article
Quantitative Proteomic and Metabolomic Profiling Reveals Altered Mitochondrial Metabolism and Folate Biosynthesis Pathways in the Aging Drosophila Eye
In Brief Hall et al. profiled the proteome, transcriptome, and metabolome of the aging Drosophila eye. The integrated analysis revealed changes in metabolism, potentially due to decreases in availability of B vitamins, together with chronic activation of immune response. - Research Article
Region-Specific Cell Membrane N-Glycome of Functional Mouse Brain Areas Revealed by nanoLC-MS Analysis
In Brief We have characterized the cell-membrane N-glycome of two major developmental divisions of the brain, the forebrain and hindbrain, and three functional derivatives from them, including the cerebral cortex, hippocampus, and cerebellum, revealing an extraordinary diversity of N-glycans expressed in a global and functional region-specific manner in the adult mouse. Furthermore, we identified +25 N-glycans able to differentiate the forebrain and hindbrain N-glycome. Additionally, over 35 N-glycans distinguished the cortex, hippocampus, and cerebellum N-glycomes and may serve as region-specific glycan biomarkers. - Research Article
Proteome Landscape of Epithelial-to-Mesenchymal Transition (EMT) of Retinal Pigment Epithelium Shares Commonalities With Malignancy-Associated EMT
In Brief EMT can play a role in retinal diseases. Here, we present a comprehensive proteomic analysis aimed at defining the temporal protein expression changes associated with EMT of stem cell–derived retinal pigment epithelial cells. Tandem mass tag and direct data-independent acquisition MS approaches were performed after inducing RPE-EMT by enzymatic dissociation. We present integration of our proteomic data with prior transcriptomic (RNA-Seq) data to provide additional insights into the RPE-EMT progression. - Research Article
Characterization of an A3G-VifHIV-1-CRL5-CBFβ Structure Using a Cross-linking Mass Spectrometry Pipeline for Integrative Modeling of Host–Pathogen Complexes
In Brief We present a pipeline that streamlines cross-linking mass spectrometry (XL-MS) data collection, data analysis, and integrative modeling of host–pathogen complexes. Using XL-MS, known atomic structures, and functional genetic data, we determined an integrative structure of the HIV-human A3G-CRL5-Vif-CBFβ complex. This structure illustrates HIV-1 Vif interaction with A3G and captures the structural dynamics and flexibility of the entire A3G-CRL5-Vif-CBFβ complex. - Research Article
Decellularization Enables Characterization and Functional Analysis of Extracellular Matrix in Planarian Regeneration
In Brief The ECM is a three-dimensional network of macromolecules that supports and regulates cell functions. Yet, only a few research organisms are available for the systematic dissection of the composition and functions of the ECM, particularly during regeneration. In this study, we report an integrative approach involving whole animal decellularization, protein mass spectrometry (LC–MS/MS), and RNA interference-based loss-of-function assays to identify both known and novel ECM components involved in regeneration. - Research Article
Characterization of R-Loop–Interacting Proteins in Embryonic Stem Cells Reveals Roles in rRNA Processing and Gene Expression
In Brief Wu et al. performed stringent purification of proteins associated with R-loops in mouse embryonic stem cells, uncovering 364 high-confidence R-loop–associated proteins. Nucleolar proteins, including numerous DEAD-box family helicase proteins, were highly enriched within this group. Closer examination of several DEAD-box helicases revealed post-transcriptional roles in production of mature rRNAs and direct or indirect roles in regulation of differentiation-associated genes. These findings reveal a vast network of R-loop–associated proteins with key functions in stem cell homeostasis. - Research Article
The Insufficient Activation of RIG-I–Like Signaling Pathway Contributes to Highly Efficient Replication of Porcine Picornaviruses in IBRS-2 Cells
In Brief Both IBRS-2 and PK-15 cells have been widely used for porcine picornavirus research. However, the virus replicates faster and causes severer CPE in IBRS-2 cells than in PK-15 cells, and the underlying mechanism remains unknown. Proteomic analyses suggested that the RLR pathway was in a dysfunctional state in IBRS-2 cells. We finally determined that the disabled signal transduction from TBK1 to IRF3 in IBRS-2 cells was the fundamental cause of dysfunction of the RLR pathway during porcine picornavirus infection. - Research Article
Global Insights Into Lysine Acylomes Reveal Crosstalk Between Lysine Acetylation and Succinylation in Streptomyces coelicolor Metabolic Pathways
In Brief The bimodification of lysine acylation has been observed in multiple organisms, while the understanding of acylation crosstalk remains at an early stage. In the current study, by implementing proteome-wide analyses, an extensive overlap of lysine acetylation and succinylation was revealed in S. coelicolor. Moreover, the modification sites were quantified by knocking out either the deacetylase ScCobB1 or the desuccinylase ScCobB2, demonstrating a possible competitive relationship between the acetylation and succinylation in vivo. Further analyses suggested that these bimodification proteins were enriched in multiple metabolic pathways including the tricarboxylic acid cycle and protein translation pathways. - Research Article
Sulfation of O-glycans on Mucin-type Proteins From Serous Ovarian Epithelial Tumors
In Brief Ovarian cancer cells secrete O-glycosylated sulfated proteins, probably with a purpose to confuse the immune system. We show that in addition to the ubiquitous sulfation on N-acetylglucosamine, we detected the less prevalent sulfation reported on galactose. Selecting three sulfotransferases that are capable of galactose sulfation, the ovarian sulfation could be mimicked recombinantly. One of these transferases (Gal3ST2) was shown to be downregulated in malignant cancer compared to benign. The data highlights that sulfation is involved in ovarian cancer development. - Research Article
Oxidative Modifications Switch Modulatory Activities of Urinary Proteins From Inhibiting to Promoting Calcium Oxalate Crystallization, Growth, and Aggregation
In Brief Previous evidence has shown that oxidative stress commonly occurs in kidney stone disease. But all the references have indicated the occurrence of oxidative stress (as the result or complication) after the stone or disease has been already developed. However, its etiologic role (as the cause) remains unknown. This first piece of evidence highlights the essential role of oxidative modifications of urinary proteins as one of the etiologies causing kidney stone formation. - Research Article
A Novel Spectral Annotation Strategy Streamlines Reporting of Mono-ADP-ribosylated Peptides Derived from Mouse Liver and Spleen in Response to IFN-γ
In Brief ADP-ribosyl peptides exhibit complex HCD-derived MS2 spectra that comprise peaks corresponding to the complete or partial dissociation of the ADP-ribosyl moiety itself (m-ions) and peaks corresponding to the residual modification on the intact peptide (P-ion) that in turn undergoes further dissociation to produce peptide fragments (p-ions). Our novel workflow annotates and scores these unique ADP-ribose fragments not recognized by standard annotation methods. This workflow increased the number of reportable ADP-ribosyl peptides identified in a mouse model for acute proinflammatory response. - Research Article
Pronounced Postmating Response in the Drosophila Female Reproductive Tract Fluid Proteome
In Brief Fertility depends on coordinated postmating processes within the female reproductive tract (FRT), including ejaculate–FRT protein interactions that regulate sperm motility, storage, and modification. Semiquantitative proteomics was utilized to characterize the FRT tissue and, separately, luminal fluid, before and after mating. The dynamic mating-induced changes in the FRT fluid inform our understanding of FRT secretory mechanisms and ejaculate–female interactions that regulate fertility. Our study highlights the utility of applying proteomic approaches to characterize the extracellular FRT environment. - Research Article
Assessing the Preanalytical Variability of Plasma and Cerebrospinal Fluid Processing and Its Effects on Inflammation-Related Protein Biomarkers
In Brief We assessed the effects of delayed sample handling on a panel of 92 inflammation-related proteins in the blood and cerebrospinal fluid. Plasma protein levels were measured at delayed centrifugation times of 1, 24, 48, and 72 h corresponding with common postal-transit delays, and changes in relative concentration were modeled and validated using an external dataset. Several proteins were selected as markers of assessing sample handling variability for application in future studies. - Research Article
Metabolic Perturbation Associated With COVID-19 Disease Severity and SARS-CoV-2 Replication
In Brief The molecular mechanisms underlying SARS-CoV-2 infection and disease severity are unclear. We used multiomics analysis in patient material and cell-line models to delineate the metabolic modulations caused by SARS-CoV-2 infection. Plasma mannose emerged as a strong biomarker for COVID-19 severity. SARS-CoV-2 depends on glutaminolysis and glycolysis for infection and replication. Infection of lung epithelial cells indicated strong metabolic adaptation with mitochondrial dysfunction. Inhibition of glycolysis and glutaminolysis limits viral production, which indicates a potential host-directed novel treatment strategy. - Research Article
Integrated Systems Analysis of the Murine and Human Pancreatic Cancer Glycomes Reveals a Tumor-Promoting Role for ST6GAL1
In Brief Pancreatic ductal adenocarcinoma is a leading cause of cancer death. Glycans are emerging as important modulators of cancer phenotypes. Herein, we used a strategic systems-based approach integrating glycomics of the KC mouse, modeling early events in transformation, with human data to identify ST6GAL1 (e.g., α-2,6-sialic acid) as a potential cancer promoter. A pancreas-specific ST6GAL1 KO in the KC mouse delayed cancer formation and reduced fibrosis. Our results highlight the importance of identifying glycans whose functions can be modeled in mice. - Research Article
A Systematic Survey of the Light/Dark-dependent Protein Degradation Events in a Model Cyanobacterium
In Brief Large-scale identification of the light/dark-regulated protein degradation events in the model cyanobacterium Synechocystis sp. PCC 6803 was conducted using quantitative proteomics. Proteins with strong degradation in light, dark, or both conditions were identified. The light-regulated degradations of multiple proteins were sensitive to photosynthetic electron transport inhibitors (DCMU and DBMIB), indicative of the redox regulation by the plastoquinone (PQ) pool in the photosynthetic electron transport chain. - Research Article
Proteogenomics Reveals Perturbed Signaling Networks in Malignant Melanoma Cells Resistant to BRAF Inhibition
In Brief Proteogenomics is a powerful tool to study the mode of action of disease-associated mutations at the genome, transcriptome, proteome, and PTM level. Here, we applied a proteogenomics workflow to study the malignant melanoma cell line A375. Such workflow, used here as a proof of concept on A375 cells, may be applicable to other cancer types, cell lines, or even patient-derived samples. - Research Article
Identification of SUMO Targets Associated With the Pluripotent State in Human Stem Cells
In Brief The role of SUMO modification in maintenance of human induced pluripotent stem cells was investigated using an inhibitor of SUMO modification. Key markers of pluripotency were lost after inhibitor treatment. Employing SUMO site proteomics, we identified 976 sites in 427 proteins. A major network of zinc-finger transcription factors linked to TRIM28 was associated with silencing of retroviral elements. At the site level there appears to be a preference for SUMO2 modification over SUMO1 in acidic domains. - Research Article
A Complex Proteomic Response of the Parasitic Nematode Anisakis simplex s.s. to Escherichia coli Lipopolysaccharide
In BriefAnisakis simplex s.s. is a seafood-borne parasite that is involved in human infections. This experimental setup mimics the coexistence of helminth and gut bacteria in the host. Describing the mechanisms of the proteomic response of A. simplex to LPS can contribute to better understanding the parasite biology and more effective treatment of anisakiasis. Our findings indicate the complexity of the proteomic response of this parasite to LPS. Obtained results are valuable in planning future strategies for studying helminths. - Research Article
Low Cell Number Proteomic Analysis Using In-Cell Protease Digests Reveals a Robust Signature for Cell Cycle State Classification
In Brief We introduce a streamlined sample processing method for bottom–up proteomics called the “in-cell digest.” Fixed cells are directly digested by trypsin to peptides for LC–MS/MS. Combined with AMPL, we analyze the proteomes of 16 unperturbed cell cycle populations using 2500 cells for each. We identify a 119-protein cell cycle signature. Using this signature, we show unbiased classification of proteomes in proteomeHD into specific cell cycle phases. Precise cell cycle classification will be important in dissecting single-cell proteome heterogeneity. - Research Article
Deep-Learning-Derived Evaluation Metrics Enable Effective Benchmarking of Computational Tools for Phosphopeptide Identification
In Brief Tandem mass spectrometry (MS/MS)-based phosphoproteomics is a powerful technology for global phosphorylation analysis. However, applying different computational pipelines to the same dataset may produce substantially different phosphopeptide identification results, underscoring a critical need for benchmarking. We present three deep-learning-derived benchmark metrics. The benchmark metrics demonstrated in this study will enable users to select computational pipelines and parameters for routine analysis of phosphoproteomics data and will offer guidance for developers to improve computational methods. - Research Article
The Circadian Clock Gene Circuit Controls Protein and Phosphoprotein Rhythms in Arabidopsis thaliana
In Brief Plants have circadian rhythms, driven by a transcription factor network. Circadian clock research has therefore focused on transcriptional regulation. However, nontranscriptional processes also play a role. Therefore, we here present circadian (phospho)proteomics time courses. We find rhythmically phosphorylated proteins with diverse biological roles and demonstrate functional relevance of one example. Most of these rhythms require the transcriptional oscillator. Moreover, most rhythmic phosphorylations peak around dawn, which is a focus of our analysis. These results increase our knowledge of nontranscriptional circadian processes. - Research Article
Quantitative Ubiquitylome Analysis Reveals the Specificity of RNF111/Arkadia E3 Ubiquitin Ligase for its Degradative Substrates SKI and SKIL/SnoN in TGF-β Signaling Pathway
In Brief In this study, we aimed to identify exhaustively the substrates of the E3 ubiquitin ligase RNF111 that activates TGF-β signaling. We performed quantitative ubiquitylome comparison of parental U2OS cells with CRISPR modified clones that express a truncated RNF111 devoid of RING domain using two approaches based on the enrichment of ubiquitylated proteins. Integrative proteomics comparison of ubiquitylome and proteome identifies SKI and SKIL as the only targets ubiquitylated and degraded by RNF111 upon TGF-β stimulation. - Research Article
Proteomic Analysis of Trichomonas vaginalis Phagolysosome, Lysosomal Targeting, and Unconventional Secretion of Cysteine Peptidases
In Brief Lysosomes represent a central degradative compartment of eukaryotes, yet little is known about biogenesis and function of this organelle in the parasitic protist Trichomonas vaginalis. We analyzed the phagolysosomal proteome that consists of over 460 proteins including important virulence factors. We demonstrated that glycosylation is involved in lysosomal protein targeting in T. vaginalis, which is unprecedented in parasitic protists. In addition to the classical secretory pathway, lysosomes are involved in unconventional protein secretion. - Research Article
The Ubiquitin Interacting Motif-Like Domain of Met4 Selectively Binds K48 Polyubiquitin Chains
In Brief Ubiquitylation is a highly complex protein modification. The diversity of the ubiquitin signal is due to the variety of different ubiquitin chains that are assembled in cells. Villamil and colleagues identified a ubiquitin binding domain with high selectivity for only one specific chain type, K48-linked polyubiquitin. They used this UIML domain to generate a K48-ubiquitin chain selective affinity reagent for proteome-wide studies of proteins modified by this specific ubiquitin chain. - Research Article
The Urinary Proteomic Profile Implicates Key Regulators for Urologic Chronic Pelvic Pain Syndrome (UCPPS): A MAPP Research Network Study
In Brief Urinary chronic pelvic pain syndrome (USPPS) is a poorly understood syndrome characterized by pelvic pain and urinary frequency and/or urgency. In this work, urinary proteomics was used to define the altered urinary proteome of UCPPS compared with healthy controls and unrelated pelvic pain diseases. The results describe the potential role of the altered extracellular matrix turnover and reflect an altered immune response. These findings may highlight novel treatment modalities and/or provide molecular targets for future diagnostic development. - Research Article
Metabolic Enzyme Alterations and Astrocyte Dysfunction in a Murine Model of Alexander Disease With Severe Reactive Gliosis
In Brief The article contains the first whole brain proteomic survey from a mouse model of Alexander disease (AxD). Several novel findings include activation of the PPAR signaling pathway, which has been reported to be protective in models of amyotrophic lateral sclerosis (ALS). Another finding related to the gliosis phenotype in AxD mice was the upregulation of fatty acid binding protein 7 (FABP7), which induces an NF-κB inflammatory response and counteracts the anti-inflammatory effects of PPAR signaling. - Research Article
Immune Checkpoint Blockade Augments Changes Within Oncolytic Virus-induced Cancer MHC-I Peptidome, Creating Novel Antitumor CD8 T Cell Reactivities
In Brief Immune checkpoint blockade augments changes within oncolytic virus-induced cancer MHC-I peptidome and contributes toward the therapy-induced novel antitumor CD8 T cell reactivities. - Research Article
Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2) Is Upregulated in Breast Epithelial–Mesenchymal Transition and Responds to Oxidative Stress
In Brief Epithelial–mesenchymal transition (EMT) is a cellular process inherent to cancer cell metastasis. Metabolic reprogramming is a driver of EMT. We performed proteomic profiling of three isogenic cell lines from human breast epithelium representing the epithelial, mesenchymal, and “partial” mesenchymal states of EMT to identify metabolic vulnerabilities associated with cell invasion. Bioinformatic and functional analysis revealed that the metabolic enzyme GFPT2 is a marker of claudin-low breast cancer, responds to oxidative stress, and impacts EMT, cell growth, and cell invasion. - Research Article
DIA-Based Proteomics Identifies IDH2 as a Targetable Regulator of Acquired Drug Resistance in Chronic Myeloid Leukemia
In Brief To understand the underlying resistance mechanisms in response to imatinib (IMA) and adriamycin (ADR), we explored two unique drug resistance models of K562 cells. We applied an optimized DIA–MS method to quantify 98,232 peptides from 7082 proteotypic proteins from these samples using four DIA software tools including OpenSWATH, Spectronaut, DIA-NN, and EncyclopeDIA. The sirtuin signaling pathway was found significantly regulated in both models, and IDH2 was identified as a druggable regulator of acquired drug resistance. - Research Article
Characterization of the AGR2 Interactome Uncovers New Players of Protein Disulfide Isomerase Network in Cancer Cells
In Brief Quantitative LC-MS/MS analysis of AGR2 interactome has identified 15 potential partners in both T47D cells and H1299 cells stably transfected with AGR2. The most interesting partners, PDIA3 and PDIA6, belong to the protein disulfide isomerase family. Stronger PDIA3 interaction with AGR2 under ER stress further supports the existence of PDI reactive network in the cells. - Research Article
Multiomic Metabolic Enrichment Network Analysis Reveals Metabolite–Protein Physical Interaction Subnetworks Altered in Cancer
In Brief Metabolism is recognized as an important driver of complex diseases, but global metabolite profiling remains a challenge. Protein expression is a poor proxy because pathway enrichment models provide an incomplete mapping between the proteome and metabolism. We developed MOMENTA, a multiomic network approach for interrogating metabolic pathways from proteomics data. Analysis of data from cancer cell lines and human tumors reveals metabolic network rewiring and oncogene connections. The metabolic networks altered in cancer are linked to clinical outcomes. - Research Article
Dichotomous Responses to Chronic Fetal Hypoxia Lead to a Predetermined Aging Phenotype
In Brief Using bottom-up proteomics and the kidney as a paradigm, we report an integrative perspective of the cellular responses to chronic fetal hypoxia, uncovering fundamental mechanisms of the fetal programming of adult diseases theory that are principally applicable to all other organs in the body. The characteristic tissue and serum biomarker profile will promote the development of novel therapeutic approaches to counteract the premature aging phenotype that seems to be associated with the fetal programming of chronic diseases. - Research Article
Temporal Analysis of Protein Ubiquitylation and Phosphorylation During Parkin-Dependent Mitophagy
In Brief We used a quantitative proteomics approach to study dynamics of the mitochondrial proteome, ubiquitylome, and phosphoproteome during early (2–6 h) and late stages (12–18 h) of mitophagy. We focused on parkin-initiated ubiquitylation and the impact on the proteome level, which pointed to an outside–in progression of ubiquitylation and protein degradation of mitochondrial subcompartments. In addition, we validated the interplay of phosphorylation and ubiquitylation on VDAC2, which directly influenced its degradation over the course of mitophagy. - Research Article
Multidimensional Dynamics of the Proteome in the Neurodegenerative and Aging Mammalian Brain
In Brief Neurodegenerative diseases are characterized by the abnormal accumulation of aggregated proteins in the brain. Using in vivo pulse isotope labeling, we screened the proteome for changes in protein turnover and abundance in multiple mouse models of neurodegeneration. These data suggest that the disease state of pathologically affected tissue is characterized by a proteome-wide increase in protein turnover and repair. In contrast, in healthy wild-type mice, aging in the mammalian brain is associated with a global slowdown in protein turnover. - Research Article
Comparative Analysis of T-Cell Spatial Proteomics and the Influence of HIV Expression
In Brief Using HIV-1 as a model virus, we compared several published analytical tools for spatial proteomics to determine the effect of viral expression. We found that when using differential centrifugation to fractionate T cells, the accuracy of classifiers was organelle dependent with variable sensitivity to viral gene expression. Identification of protein translocations by the BANDLE pipeline showed the highest agreement with known HIV interactors and targets. These findings lay a foundation for future spatial proteomics studies of viral infection and expression. - Research Article
Interactome Analysis of Human Phospholipase D and Phosphatidic Acid-Associated Protein Network
In Brief Using a proteomic approach, Kattan et al. defined the protein interaction network for the human phospholipase D family of enzymes and their lipid product phosphatidic acid and revealed diverse cellular signaling events involving this important lipid metabolic pathway. - Research Article
Integrated Application of Multiomics Strategies Provides Insights Into the Environmental Hypoxia Response in Pelteobagrus vachelli Muscle
In Brief Aquatic ecosystems are increasingly stressed because of nutrient enrichment, pollutants, and global warming, which have seriously depleted oxygen concentrations. This sudden and significant lack of oxygen has resulted in persistent increase in fish mortality rates. Studying the molecular mechanisms involved in hypoxia adaptation in fishes will help researchers understand fish speciation and the evolution of the hypoxia-signaling pathway and guide the breeding of hypoxia-tolerant fish strains. - Research Article
Quantitative Metaproteomics and Activity-based Protein Profiling of Patient Fecal Microbiome Identifies Host and Microbial Serine-type Endopeptidase Activity Associated With Ulcerative Colitis
In Brief Thuy-Boun et al. quantitatively compare the stool microbiomes of healthy and ulcerative colitis patients with label-free data-dependent LC-MS/MS proteomics. Their analyses identified 176 significantly enriched protein groups between the two cohorts, and serine-type endopeptidase activity was one such functionality overrepresented in UC patients. Pre-enrichment of the clinical samples with a biotinylated fluorophosphonate probe further demonstrated that serine endopeptidases are active within the patient fecal samples and that additional putative serine hydrolases were identified by this approach compared with unenriched profiling. - Research Article
A Dynamic and Combinatorial Histone Code Drives Malaria Parasite Asexual and Sexual Development
In Brief The complex combinatorial histone code of the malaria parasite is revealed through advanced, quantitative middle-down proteomics. Cross talk between histone PTMs (including novel PTMs) is dynamic and stage specific and includes arginine methylation. Chromatin proteomics show that the transcription factor AP2-G2 interacts with the combination of H3K18acK23ac to drive mature gametocyte transmissibility. The connectivity of the histone code in these parasites ultimately points toward a higher order of gene regulation involved in essential development processes than previously thought. - Research Article
Proteomics Profiling of Human Synovial Fluid Suggests Increased Protein Interplay in Early-Osteoarthritis (OA) That Is Lost in Late-Stage OA
In Brief This study presents data of the proteome in human synovial fluid from three groups representing early-stage knee OA, end-stage knee OA, and controls without knee OA. The early stage had an increased protein activity, while in end-stage OA, there was a loss of interplay between the proteins. These results highlight the importance of studying the early stage of OA progression and that the interplay between the proteins may be an additional key element in disentangling the complex OA pathogenesis. - Research Article
Mass Spectrometric and Glycan Microarray–Based Characterization of the Filarial Nematode Brugia malayi Glycome Reveals Anionic and Zwitterionic Glycan Antigens
In Brief While parasite glycans form the basis of highly successful diagnostic assays, filarial glycosylation is largely unexplored. Therefore, we conducted a comprehensive structural characterization of N-linked and GSL glycans of Brugia malayi. Our work revealed anionic and zwitterionic glycan motifs as major antibody targets. Glycan microarray analysis showed the induction of IgG and IgM to these glycans in a rhesus macaque infection model as well as a specific IgG response associated with infection in individuals from a Brugia malayi endemic area. - Research Article
Multiattribute Glycan Identification and FDR Control for Glycoproteomics
In Brief Glycoproteomics has seen rapid advances in methods for identifying glycopeptides, but challenges remain confidently determining the composition and structure of the attached glycan. We have developed a method using multiple sources of information from the mass spectrum to assign the composition of N-linked glycopeptides and an associated method for false discovery rate control. We show that this method is able to identify more glycopeptide spectra while also providing more accurate composition assignments than existing tools. - Research Article
Simple But Efficacious Enrichment of Integral Membrane Proteins and Their Interactions for In-Depth Membrane Proteomics
In Brief Membrane proteins, particularly integral membrane proteins, are barely detected in bottom–up proteomics because of their complex nature and abundant soluble proteins. We applied standard biochemical procedures to optimize the sample preparation method for membrane proteome. Membranes were precipitated by ultracentrifugation, followed by treatment with urea or alkaline solutions to remove contaminants. This enrichment was critical to obtain comprehensive membrane proteome data. Among the methods, washing membranes by urea distinctly revealed intricate membrane proteome with keeping protein–protein interactions. - Research Article
Integrated Liver and Plasma Proteomics in Obese Mice Reveals Complex Metabolic Regulation
In Brief Obesity leads to the development of type 2 diabetes and nonalcoholic fatty liver disease. To identify the underlying processes of obesity-induced metabolic dysfunction, we performed proteomics in liver and plasma of ob/ob mice. Peroxisomal biogenesis and dysregulation of the secretory machinery were apparent in the liver of obese mice, alongside substantial alterations to the plasma proteome. Integration of these datasets identified putatively liver-derived proteins that are systemically dysregulated in obesity, many of which are also altered in human metabolic diseases. - Research Article
Proteomics Uncovers Novel Components of an Interactive Protein Network Supporting RNA Export in Trypanosomes
In Brief This work was executed under full ethical compliance, and authorship is limited to those who have made significant contributions. The manuscript is original work, and works of others have been appropriately cited. We provide raw data for appropriate datasets at public databases. This work was performed standard compliant with community-acceptable guidelines and parameters. No known hazard was caused, nor any involvement of human subjects. Animal use for production of antibodies was performed under the institutional ethical guidelines. - Research Article
Multiple Reaction Monitoring-Mass Spectrometry Enables Robust Quantitation of Plasma Proteins Regardless of Whole Blood Processing Delays That May Occur in the Clinic
In Brief In the clinic, delays between blood collection and plasma generation are often unavoidable, possibly impacting intact protein-assay measurements, such as ELISA. Here we investigated the impact of plasma processing delays (0 to 40 h) on peptide-centric protein quantitation via validated LC/MRM-MS assays. From 159 LC/MRM-MS assays, 139 were ‘stable’ (RSD < 20%), 14 ‘semistable’ (RSD 20–30%), and 6 ‘unstable’ (RSD > 30%), demonstrating robustness and thus the potential for plasma-protein quantitation by validated LC/MRM-MS assays in a clinical setting. - Research Article
Characterization of Glycoproteoforms of Integrins α2 and β1 in Megakaryocytes in the Occurrence of JAK2V617F Mutation-Induced Primary Myelofibrosis
In Brief Changes in glycosylation were documented at multiple sites in integrins β1 and α2 isolated from bone marrow of WT and myelofibrotic (JAK2V617F) mouse megakaryocytes. Glycopeptiforms at 11 out of the 12 potential N-glycosylation sites of integrin β1 and at all nine potential glycosylation sites on integrin α2 were characterized. - Research Article
Precision Glycoproteomics Reveals Distinctive N-Glycosylation in Human Spermatozoa
In Brief An in-depth N-glycoproteome map of human spermatozoa has been established by interpreting precise glycan structures at each glycosite, which allowed revealing a number of distinctive glycoproteins and heavily fucosylated glycans in spermatozoa. Sialylation and Lewis epitopes were enriched in the biological process of immune response, and highly expressed bisected core structure and LacdiNAc were detected in spermatozoa acrosome. These findings lay the foundation for subsequent functional studies of glycosylation and glycan structures in spermatozoa and male reproduction diseases. - Research Article
The Acyl-Proteome of Syntrophus aciditrophicus Reveals Metabolic Relationships in Benzoate Degradation
In BriefSyntrophus aciditrophicus is a syntrophic bacterium degrading fatty and aromatic acids into acetate, CO2, formate, and H2, consumed by methanogenic archaea. The syntroph’s acyl-lysine modifications were analyzed with a workflow avoiding antibody enrichment, enabling unbiased global acylation profiling. Seven acyl modification types were identified, six corresponding to reactive acyl-CoA species intermediates in benzoate degradation. Benzoate-degrading enzymes were also prominent among the 60 acylated proteins. The abundant acylations and active deacylases suggest that post-translational modifications directly regulate syntrophic benzoate degradation. - Research Article
In-Depth Matrisome and Glycoproteomic Analysis of Human Brain Glioblastoma Versus Control Tissue
In Brief Glioblastoma (GBM) is the most malignant human brain tumor. It is critical to understand the molecular mechanisms in GBM and identify clinical markers. Extracellular matrix or matrisome is implicated in important neurological processes in brain cancer. But less attention has been paid to matrisomal changes and related glycosylation in GBM. In this manuscript, we performed an in-depth matrisomal, glycomic, and glycoproteomic analysis of control and GBM samples to identify key alterations in matrisomal components, strengthening our knowledge of GBM pathology. - Research Article
Hypoxia Is a Dominant Remodeler of the Effector T Cell Surface Proteome Relative to Activation and Regulatory T Cell Suppression
In Brief The tumor microenvironment (TME) features immunosuppressive regulatory T cells (Tregs) and areas of hypoxia. Given the importance of surface proteins in T cell antitumor function, we performed quantitative cell surface proteomics to determine how these factors affect the primary effector T cell surface proteome (surfaceome). We discovered that Treg coculture and hypoxia reduced expression of nutrient transporters, among other proteins implicated in T cell activation. Together, our findings reveal insights into how these immunosuppressive factors modulate the T cell surfaceome. - Research Article
SP3-Enabled Rapid and High Coverage Chemoproteomic Identification of Cell-State–Dependent Redox-Sensitive Cysteines
In brief Oxidative stress has been implicated in most biological processes. To improve our understanding of how cells are impacted by and respond to oxidative stressors, we developed a new proteomics method, SP3-Rox that can accurately quantify the oxidation state of thousands of cysteines proteome-wide. We establish the MSFragger algorithm as a computational platform capable of quantifying changes to thiol oxidation, including for peptides containing multiple cysteines. Application of SP3-Rox uncovered cell-state–dependent redox-sensitive residues in primary human T cells. - Research Article
Proteogenomic Analysis of Breast Cancer Transcriptomic and Proteomic Data, Using De Novo Transcript Assembly: Genome-Wide Identification of Novel Peptides and Clinical Implications
In Brief Alternative splicing of known protein-coding genes and expression of noncoding sequences of the human genome are increasingly expanding the functional diversity of proteins. These events may be specific to cell type or physiological condition of the cells and may be deregulated in cancer. Using proteogenomic analysis, we have identified novel peptides in breast cancer that arise from such events. High expression of some of them is associated with patients' survival and may be studied as prognostic indicators.
