Research Articles
- 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
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
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
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
Unraveling the MAX2 Protein Network in Arabidopsis thaliana: Identification of the Protein Phosphatase PAPP5 as a Novel MAX2 Interactor
In Brief The F-box protein MAX2 is a central component in the signaling pathway of the phytohormones, strigolactones and smoke-derived karrikins. These molecules are perceived by the related receptors D14 and KAI2, respectively. Here, we used tandem and single affinity purifications to broaden the MAX2, KAI2, and D14 interaction network and identified PAPP5 as a novel player. PAPP5 might act via dephosphorylation of MAX2 to modulate KAI2-dependent seed germination and young seedling phenotypes. - Research Article
Prediction and Validation of Mouse Meiosis-Essential Genes Based on Spermatogenesis Proteome Dynamics
In Brief A comprehensive proteomic characterization of the germ cells at different meiotic stages of spermatogenesis was investigated, the dynamical abundances of a total of 8002 proteins formed four proteomic clusters across these meiotic substages, offering novel insights into meiotic protein expression. A model of supervised machine learning based on the mouse genomics informatics and the spermatogenesis proteomic dataset was established to predict meiosis-essential genes, while the prediction was experimentally verified by knockout mice. - Research Article
Identification of Novel Serological Autoantibodies in Takayasu Arteritis Patients Using HuProt Arrays
In Brief A two-phase approach was adopted to identify novel autoantibody biomarkers of Takayasu arteritis (TAK) patients. HuProt array together with a TAK-focused array identified eight novel autoantibodies as TAK-specific biomarker candidates. Of them, the anti-SPATA7, -QDPR, and -PRH2 have the potential to be readily adopted in a clinical setting. - Research Article
Systematic Proteome and Lysine Succinylome Analysis Reveals Enhanced Cell Migration by Hyposuccinylation in Esophageal Squamous Cell Carcinoma
In Brief The proteome, phosphorylome, lysine acetylome, and succinylome were quantified for esophageal squamous cell carcinoma (ESCC) and matched control cells. Our results identify hyposuccinylation in cancer cells and reveal that ESCC malignant behaviors are inhibited once the level of succinylation was restored in vitro or in vivo, which is further confirmed in primary ESCC specimens. Our findings demonstrate that lysine succinylation may alter ESCC metabolism and migration, providing a new insight into the functional significance of posttranslational modification in cancer biology. - Research Article
Multisample Mass Spectrometry-Based Approach for Discovering Injury Markers in Chronic Kidney Disease
In Brief The key renal-expressed proteins including protein S and galectin-1 associated with CKD stages were determined by applying quantitative MS-based proteomics using multiple types of samples. The discovered proteins represent potential markers of chronic kidney injury related to renal hypoxia and candidate contributors to CKD pathophysiology. - Research Article
Transcriptome and Secretome Analysis of Intra-Mammalian Life-Stages of Calicophoron daubneyi Reveals Adaptation to a Unique Host Environment
In brief Paramphistomosis, caused by Calicophoron daubneyi, is a parasitic infection of ruminant livestock currently spreading throughout Western Europe. Here, transcriptome analysis of four intramammalian life-cycle stages, integrated with secretome profiling of the infective stage and adult parasites (responsible for acute and chronic diseases, respectively), revealed how selected families of unique virulence factors and immunomodulators are regulated in accordance with fluke development and migration. This analysis allowed the development of the first coproantigen-based ELISA for paramphistomosis. - Research Article
CIITA-Transduced Glioblastoma Cells Uncover a Rich Repertoire of Clinically Relevant Tumor-Associated HLA-II Antigens
In Brief CD4+ T cell responses are crucial for inducing and maintaining effective anticancer immunity; however, in glioblastoma and many solid tumors, HLA-II complexes are hardly ever naturally expressed. Hence, little is known about immunogenic HLA-II epitopes in glioblastoma. With stable expression of the class II major histocompatibility complex transactivator (CIITA) coupled to a detailed immunopeptidomics, we uncovered a remarkable breadth of the HLA-ligandome in three glioblastoma cell lines and identified many cancer-associated ligands with implications for the development of cancer immunotherapies. - Research Article
N-Glycomic Signature of Stage II Colorectal Cancer and Its Association With the Tumor Microenvironment
In Brief There is a need for better stratification of patients with stage II colorectal cancer as the treatment option remains controversial. Using MS imaging, we have characterized the N-glycosylation signatures of the different cell populations in stage II CRC tissues. We found, looking at the tumor microenvironment, that the cancer N-glycosylation signature spreads into the surrounding stroma at the invasive front of the tumor. This finding was more outspoken in patients with a worse outcome. - Research Article
In-Depth Characterization of the Staphylococcus aureus Phosphoproteome Reveals New Targets of Stk1
In Brief An optimized workflow for phosphopeptide enrichment of Gram-positive bacteria has been developed, revealing extensive protein phosphorylation in S. aureus. Using mutant strains and quantitative proteomics, 74 downstream targets of the kinase Stk1 were identified in S. aureus. Given the total number of 3800 identified phosphosites, other, yet unannotated, kinases are expected to exist in S. aureus. - Research Article
Separation and Identification of Permethylated Glycan Isomers by Reversed Phase NanoLC-NSI-MSn
In Brief Structural analysis of glycoprotein N-linked and O-linked glycans is greatly enhanced by permethylation following release from the protein backbone. HPLC of permethylated glycans resolves isomeric glycans that are difficult to discern by direct infusion methodologies. An approach is described that utilizes standard peptide separation columns and accessible instrumentation. The methodology is easily adoptable by laboratories already familiar with LC-based peptide separations to achieve isomeric resolution of glycans released from biological materials. - Research Article
Site-specific N-glycosylation Characterization of Recombinant SARS-CoV-2 Spike Proteins
In Brief Site-specific N-glycosylation of SARS-CoV-2 spike proteins expressed in human and insect cells has been decoded via enriched glycopeptides using a stepped collision energy HCD scan. The regular N-glycan occupancy on preserved N-glycosites has been revealed, despite the heterogeneity in N-glycan compositions. The complex N-glycans on the human cell-expressed spike protein reflect the relatively accessible antigens and epitopes for immune recognition. The insect cell-expressed spike protein subunits decorated with immature high-mannose N-glycans might become promising recombinant vaccine candidates. - Research Article
Interspecies Differences in Proteome Turnover Kinetics Are Correlated With Life Spans and Energetic Demands
In Brief A proteomic approach was used to measure protein turnover rates within fibroblasts isolated from species with diverse life spans. We found that life span is negatively correlated with turnover rates of highly abundant proteins. Despite having slower rates of protein turnover, long-lived naked mole rat cells tolerate protein misfolding stress more effectively than short-lived mouse cells. We suggest that in lieu of rapid constitutive turnover, long-lived species have evolved more energetically efficient mechanisms for selective detection and clearance of damaged proteins. - Research Article
A Potential Role for the Gsdf–eEF1α Complex in Inhibiting Germ Cell Proliferation: A Protein-Interaction Analysis in Medaka (Oryzias latipes) From a Proteomics Perspective
In Briefgsdf is a unique TGFα essential for testicular differentiation in medaka (Oryzias latipes). We used a His-tag “pull-down” assay and a yeast 2-hybrid (Y2H) screening to identify a Gsdf–eEF1α complex in the adult testis. Physical interaction of Gsdf and eEF1α was confirmed by a paired Y2H assay and coimmunoprecipitation in the adult testis. Results of proteomics analysis (PXD022153) and ultrastructural observations support the potential role of the Gsdf–eEF1α complex in inhibiting germ cell proliferation, which may be conserved in vertebrates. - Research Article
The Complexity and Dynamics of the Tissue Glycoproteome Associated With Prostate Cancer Progression
In Brief Integrated glycomics and glycoproteomics with reference to the established Tissue Atlas were used to uncover the cell-, protein-, site-, and structure-specific N- and O-glycosylation in prostate cancer tissues spanning five disease grades relatively to benign prostatic hyperplasia control tissues. Dynamic cell-specific changes in the paucimannosylation and oligomannosylation of known bone marrow– and prostate-derived glycoproteins, respectively, and increased N-glycan branching and core 2-type O-glycosylation of known extracellular matrix glycoproteins were found to be key changes associated with prostate cancer progression. - Research Article
Plasma Proteomes Can Be Reidentifiable and Potentially Contain Personally Sensitive and Incidental Findings
In Brief Due to its unbiased nature, proteomics may raise ethical and regulatory concerns. Plasma proteome samples can be reidentified given genomic information. Furthermore, plasma proteomes contain information of potential sensitive nature. Incidental findings can help diagnose unrelated but actionable disease states. - Research Article
Carboxylate-Modified Magnetic Bead (CMMB)-Based Isopropanol Gradient Peptide Fractionation (CIF) Enables Rapid and Robust Off-Line Peptide Mixture Fractionation in Bottom-Up Proteomics
In Brief We propose a novel method for fractionating tryptic peptide mixtures on carboxylate-coated magnetic beads. It is an extension of the previously reported SP3 (single-pot solid phase–enhanced sample preparation) protein and peptide cleanup method and provides an effective but complementary approach to other commonly used fractionation methods including strong cation exchange (SCX) and reversed phase (RP)-based chromatography. - Research Article
The Mechanism of NEDD8 Activation of CUL5 Ubiquitin E3 Ligases
In Brief Hydrogen-deuterium exchange mass spectrometry (HDX-MS) reveals how a CUL5 E3 ligase is activated for polyubiquitylation of its substrate. - Research Article
Systematic Identification of Plasmodium Falciparum Sporozoite Membrane Protein Interactions Reveals an Essential Role for the p24 Complex in Host Infection
In Brief Malaria remains one of the world's most deadly infectious diseases, and host infection is initiated when the sporozoite form of the P. falciparum parasite is transmitted by the bite of an infected mosquito. Here, we compile a library of soluble recombinant sporozoite cell surface and secreted proteins and test them for direct interactions using an extracellular protein interaction assay. We identify three conserved complexes and show that one, the p24 complex, is essential for host liver-stage infection. - Research Article
High-Throughput and Site-Specific N-Glycosylation Analysis of Human Alpha-1-Acid Glycoprotein Offers a Great Potential for New Biomarker Discovery
In Brief For the first time, a cost-effective method for a high-throughput detailed AGP N-glycosylation profiling was developed, which includes site-specific glycosylation information. Using the method, it was demonstrated that AGP N-glycan profile is stable in a healthy individual. Furthermore, using the method on a pilot cohort, it was found that site-specific AGP N-glycan profile could help distinguish individuals who are at risk of type 2 diabetes. The method presents a new valuable tool for investigation of AGP’s large biomarker potential. - Research Article
Quantitative Data-Independent Acquisition Glycoproteomics of Sparkling Wine
In Brief Glycoproteins in sparkling wine are especially important for its sensory properties. Here, we developed a novel quantitative glycoproteomic approach to investigate the glycoproteomes of sparkling wine. The protein- and glycopeptide-centric approaches enabled robust quantification from small volumes of sparkling wine and highlighted key changes that may contribute to sparkling wine quality. - Research Article
Thermal Proteome Profiling in Zebrafish Reveals Effects of Napabucasin on Retinoic Acid Metabolism
In Brief An optimized protocol for thermal proteome profiling in zebrafish embryo lysates has been developed. Using this protocol, it was shown that the Stat3 signaling inhibitor napabucasin stabilizes several aldehyde dehydrogenases. ALDH activity was activated by napabucasin in vitro and affected the retinoic acid pathway in vivo. These results show that thermal proteome profiling in whole zebrafish embryo lysate facilitates direct correlation of in vivo effects of small molecule drugs with their protein targets. - Research Article
Integrated Glycoproteomics Identifies a Role of N-Glycosylation and Galectin-1 on Myogenesis and Muscle Development
In Brief The proteome, N-glycome, and N-glycoproteome have been determined during myotube formation. This revealed a complex regulation of N-glycosylation including a switch in sialylation linkages, digalactosylation, and paucimannosylation on receptors and adhesion molecules. The data also revealed regulation of several glycan-binding proteins including LGALS1, and functional validation revealed a role of this protein in myogenesis and skeletal muscle development. - Research Article
Proteogenomic Characterization of the Pathogenic Fungus Aspergillus flavus Reveals Novel Genes Involved in Aflatoxin Production
In BriefA. flavus is a pathogenic fungus capable of producing aflatoxin, which is harmful and carcinogenic to animals and human. In this study, we presented the first comprehensive draft map of the A. flavus proteome and a wide range of posttranslational modifications. This is the first study we are aware of to have employed such a proteomic approach to improve Aspergillus genomic annotation, which will serve as a valuable resource for future efforts to explore the synthesis and excretion of aflatoxins. - Research Article
Imaging Mass Spectrometry and Lectin Analysis of N-Linked Glycans in Carbohydrate Antigen–Defined Pancreatic Cancer Tissues
In Brief N-glycosylation is an attractive target for PDAC biomarker discovery because of its well-understood roles in oncogenesis, cancer maintenance, and metastasis. Using MALDI-IMS, we observed distinct histopathology localized differences in N-glycosylation distributions between healthy and cancerous tissues. We combined the tumor-to-normal ratio of N-glycan changes determined from IMS with biomarker IHC data into modeling which improved PDAC identification over models utilizing either data set individually. This multiplexed approach potentiates the development of cross-disciplinary biomarker panels for pancreatic cancer detection. - Research Article
Peak Filtering, Peak Annotation, and Wildcard Search for Glycoproteomics
In Brief New features have been added to popular commercial software. The features are demonstrated on several publicly available data sets and are used to discover sulfated N-glycans on Fc gamma receptor IIIa, O-linked glycosylation on rat neuropeptides, and prenylated cysteines in human cells. - Research Article
Quantitative Proteomics Reveals Association of Neuron Projection Development Genes ARF4, KIF5B, and RAB8A With Hirschsprung Disease
In Brief Quantitative proteomics revealed differentially expressed proteins in paired colon tissues from 21 patients diagnosed with Hirschsprung disease (HSCR). Hierarchical clustering suggests that the protein abundancy and expression pattern in normal intestinal tissues are closely related to gender. Gene ontology enrichment analysis revealed spliceosome, axon guidance, and extracellular exosome as the most related biological processes/molecular functions. Three neuron projection development molecules, ARF4, KIF5B, and RAB8A, were downregulated in the aganglionic segment which may be involved in the pathogenesis of HSCR. - Research Article
Proteome Analysis Reveals a Significant Host-Specific Response in Rhizobium leguminosarum bv. viciae Endosymbiotic Cells
In Brief Comparison of proteomes from nodule bacteroids produced by a single Rhizobium leguminosarum strain in two different hosts (pea and lentil) reveals the existence of dozens of host-specific rhizobial proteins in the endosymbiotic cells, thus suggesting the requirement of rhizobia to adapt to different cellular environments. Host-specific sets of plant-derived nodule-specific cysteine-rich peptides were also found in the bacteroids. - Research Article
Peptidomics-Driven Strategy Reveals Peptides and Predicted Proteases Associated With Oral Cancer Prognosis
In Brief Mass spectrometry–based peptidomics has been applied to the saliva of patients with oral squamous cell carcinoma, enabling the discovery of signatures correlated with poor prognostic factors, including lymph-node metastasis. Sequence analysis of differential peptides allowed the prediction of proteases implicated in endogenous proteolysis and associated with prognosis in head and neck cancers. The results of combined saliva peptidomics and proteomics revealed that the accentuated proteolysis in patients with oral squamous cell carcinoma and lymph-node metastasis concurs with reduced levels of protease inhibitors in saliva. - Research Article
Proteomic Identification of Coxiella burnetii Effector Proteins Targeted to the Host Cell Mitochondria During Infection
In Brief A broad, unbiased proteomics-based screen with organelle purification to study the host–pathogen interactions occurring between the host cell mitochondrion and the Q fever pathogen Coxiella burnetii. This reveals a subset of Coxiella effector proteins at mitochondria during infection. Our study adapts high-sensitivity proteomics, providing a robust strategy to examine the subcellular localization of effector proteins during native infection. - Research Article
On the Robustness of Graph-Based Clustering to Random Network Alterations
In Brief Networks are an important way to represent the connections within a system, and it is useful to identify tightly connected clusters within a network (e.g., protein complexes). However, biological networks often have noise, and our results demonstrate that clustering is sensitive to noise at levels typical of biological networks (e.g., 10–50%). We developed an R-based tool (clust.perturb) that can predict the real-world reproducibility of individual clusters for any clustering algorithm by randomly perturbing the network. - Research Article
Protein Modification Characteristics of the Malaria Parasite Plasmodium falciparum and the Infected Erythrocytes
In Brief Tandem mass tag labeling, MS-based proteomics, and posttranslational modification (PTM)-omics were employed to identify samples of Plasmodium falciparum and its host erythrocytes at six time points, with healthy erythrocytes as controls. Dynamic modification levels of six PTM-omics (phosphorylation, acetylation, crotonylation, 2-hydroxyisobutyrylation, N-glycosylation, and ubiquitination) were comprehensively analyzed. Our research involved in the biology of malaria parasites from the overall PTM-omics level, laying an important foundation for pathogenic biology, cell biology, and drug mining of new antimalarial drugs.
