Research Articles
- Research Article
Direct Proteomic Detection and Prioritization of 19 Onchocerciasis Biomarker Candidates in Humans
In BriefOnchocerca volvulus infects over 20 million people, causing severe disability. Diagnostic tests cannot reliably detect live adult worms, which is critical for disease elimination efforts. Plasma and urine samples from O. volvulus-infected individuals (and uninfected controls) were analyzed by MS proteomics to directly identify O. volvulus proteins in hosts. A total of 19 proteins were prioritized. This set of biomarker candidates is a valuable resource to further explore for the diagnosis of active O. volvulus infections. - Research Article
SKAP2 Modular Organization Differently Recognizes SRC Kinases Depending on Their Activation Status and Localization
In Brief SRC kinases play multiple functions in different metabolic pathways. To perform each of these functions, they interact specifically with other proteins. This work describes how the SKAP2 protein, an assembly platform, interacts with these kinases to better understand their combined action. New and more specific therapies could be developed from this type of work, particularly in oncology. - Research Article
Mass Spectrometry–Based Proteomics Analysis of Human Substantia Nigra From Parkinson's Disease Patients Identifies Multiple Pathways Potentially Involved in the Disease
In Brief We conducted an in-depth proteome analysis of human substantia nigra tissues from 15 Parkinson’s disease (PD) patients and 15 healthy control individuals to uncover dysregulated pathways in PD. We identified 10,040 proteins with 1140 differentially expressed proteins in the substantia nigra of PD patients, discovering mitoribosome proteins were the most dysregulated proteins, followed by ribosome, RNA splicing, and complement proteins. This study has discovered that mitoribosome dysfunction is potentially involved in the PD pathogenesis process for the first time. - Research Article
Proximity Labeling Facilitates Defining the Proteome Neighborhood of Photosystem II Oxygen Evolution Complex in a Model Cyanobacterium
In Brief An effective workflow for APEX2-based proximity labeling in the model cyanobacterium Synechocystis sp. PCC 6803 was developed. Proximity labeling of proteins proximal to the photosystem II oxygen evolution complex by PsbO-APEX2 resulted in proteomic identification of 38 integral thylakoid membrane proteins and 93 luminal proteins, thereby defining the proteome neighborhood of photosystem II in the luminal side. - Research Article
Multiple Layers of Complexity in O-Glycosylation Illustrated With the Urinary Glycoproteome
In Brief In this study, mass spectrometric data acquired on urinary O-glycopeptides were analyzed by four software packages. The results were compared, and the rate of misidentification was assessed. The major factors leading to data misinterpretation were identified, and software development suggestions aiming more reliable automated data interpretation were made. - Research Article
A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer–Associated Stellate Cell Small Extracellular Vesicles
In Brief In this article, we report that cancer-associated HPSC cells secreted more sEVs than primary stellate cells (HPaStec) and had no significant growth effects on the cancer cells. Intact membrane-associated proteins may be essential for sufficient uptake of sEVs by both normal and cancer cells. Comparative proteomics analysis between the 2 different sEVs revealed differentially expressed proteins. sEVs could potentially be targeted as a cargo vehicle for the safe delivery of drugs or other biological materials to the cancer cells. - Research Article
Red Blood Cells Protein Profile Is Modified in Breast Cancer Patients
In Brief This study demonstrates for the first time that the protein composition of red blood cells is different in the presence of a breast tumor, providing new information on the potential role of RBCs in the context of disseminated disease. In addition, it points to the value of red blood cell proteins in the prognosis and diagnosis of metastatic breast cancer in a non-invasive way. - Research Article
Glycoproteomics Landscape of Asymptomatic and Symptomatic Human Alzheimer’s Disease Brain
In Brief Glycosylation of proteins in human brains, especially those with Alzheimer’s disease (AD) is not well-known. We employed multi-lectin enrichment, HILIC, and mass spectrometry–based proteomics to profile N-glycoproteins in normal, asymptomatic, and symptomatic AD brains. Unlike other organs, the brain consists of mostly high-mannosidic glycans, notably the Man5 N-glycans and complex N-glycans that are fucosylated and bisected. We observed site-specific differences in glycosylation among different AD stages, such as the number of antennae or the frequency of fucosylation. - Research Article
Global Phosphoproteomics Unveils Kinase-Regulated Networks in Systemic Lupus Erythematosus
In Brief We established the global phosphoproteome landscape of peripheral blood mononuclear cells from healthy controls, systemic lupus erythematosus (SLE) patients (SLE_S, SLE_A), and rheumatoid arthritis by combining high-throughput phosphoproteomics with bioinformatics approaches. We validated some of the key phosphosites and kinases and then built kinase-regulated networks for SLE. The map of the global phosphoproteomic enables further understanding of the pathogenesis of this disease and will provide strategies for seeking potential therapeutic targets for SLE. - Research Article
Regio-Specific N-Glycome and N-Glycoproteome Map of the Elderly Human Brain With and Without Alzheimer’s Disease
In Brief Glycosylation plays an important role in brain development and function. The cell membrane–associated N-glycans and glycoproteins from the human brain were characterized using a nanoflow LC–MS/MS platform. The brain regions included the frontal, temporal, parietal, occipital, cingulate, lateral cerebellar and orbitofrontal cortex, posterior hippocampus, thalamus, caudate nucleus, and pons. We illustrate the utility of glycomic and glycoproteomic tools to define the general breadth and depth in glycans associated with the elderly human brain and some neuropathological conditions. - Research Article
In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates
In Brief An optimized workflow for phosphopeptide enrichment has been developed in Clostridioides difficile revealing extensive protein phosphorylation. Using kinase or phosphatase mutant strains and quantitative proteomics, 41 and 114 downstream targets of the kinases PrkC and CD2148, respectively, were identified in C. difficile. We confirmed the phosphorylation of PrkC and Spo0A in vitro and of FtsK in vivo. - Research Article
Proteomic Profiling of Intra-Islet Features Reveals Substructure-Specific Protein Signatures
In Brief Improved sample handling techniques have enabled us to go from studying the whole pancreas, to isolated whole islets, to islet sections, and now to islet substructures isolated from within islet sections. Using a microfluidic nanodroplet-based proteomics platform coupled with laser capture microdissection and field asymmetric waveform ion mobility spectrometry, we present an in-depth investigation of protein profiles from intra-islet features. The protein coverage and expression profiles obtained are sufficient to fully distinguish each of these features while presenting their own unique proteome signatures. - Research Article
Open Modification Searching of SARS-CoV-2–Human Protein Interaction Data Reveals Novel Viral Modification Sites
In Brief Public MS data were reanalyzed using open modification searching to investigate the presence of post-translational modifications during interaction of viral severe acute respiratory syndrome coronavirus 2 proteins and human host proteins. We examined several post-translational modifications, including phosphorylation, ubiquitination, and S-nitrosylation, in the context of the virus–host protein–protein interaction network to obtain novel hypotheses about the functional roles of these modifications during viral infection by severe acute respiratory syndrome coronavirus 2. - Research Article
Soluble ANPEP Released From Human Astrocytes as a Positive Regulator of Microglial Activation and Neuroinflammation: Brain Renin–Angiotensin System in Astrocyte–Microglia Crosstalk
In Brief Understanding the astrocyte secretome may provide insights into the regulatory mechanisms of neuroinflammation through astrocyte-mediated communication. In this study, we found that human astrocytes release the soluble form of aminopeptidase N as a component of the brain renin–angiotensin system, positively regulating microglial activation. Our findings suggest the diagnostic and therapeutic potential of soluble form of aminopeptidase N in neuroinflammatory diseases. - Research Article
Probing the sORF-Encoded Peptides of Deinococcus radiodurans in Response to Extreme Stress
In Brief To investigate the SEPs related to extreme stress, the integrated workflow consisting of multiple omics approaches were streamlined. The SEPome of Deinococcus radiodurans containing 109 novel SEPs was described. Among them, SEP068184 exhibited negative regulation of oxidative stress resistance in a comparative phenotypic assay of its mutants. Collectively, the SEPome dataset provides a rich resource for research on the molecular mechanisms of the response to extreme stress in organisms. - Research Article
A Complex Connection Between the Diversity of Human Gastric Mucin O-Glycans, Helicobacter pylori Binding, Helicobacter Infection and Fucosylation
In BriefHelicobacter pylori colonizes the gastric mucus layer of half the world population. Mucin glycans in this mucus layer govern pathogen virulence, growth, and adhesion. The 650 O-glycan structures identified in this study represent a reliable database of structures that are of putative importance for host–pathogen interactions. We further showed that both the enormous gastric O-glycan diversity and avidity for H. pylori are governed by fucosylation and infection-associated O-glycans. - Research Article
A New Monoclonal Antibody Enables BAR Analysis of Subcellular Importin β1 Interactomes
In Brief Proximity biotinylation with a new monoclonal antibody reveals subcellular importin β1 interactomes. - Research Article
The Mechano-Ubiquitinome of Articular Cartilage: Differential Ubiquitination and Activation of a Group of ER-Associated DUBs and ER Stress Regulators
In Brief Using ubiquitomics, we identified a unique ubiquitin signature of articular cartilage upon mechanical injury. A set of deubiquitinases (DUBs) and ER stress regulators including YOD1, ATXN3, RAD23B, VCP/p97, and Ubiquilin 1 were differentially ubiquitinated and their ubiquitination status was linked to enhanced DUB activity and upregulated ER stress response following injury. We observed a rapid increase in global DUB activity in injured and in human osteoarthritic cartilage. Data implicates DUBs as potential targets to modulate injury-induced tissue damage in osteoarthritis. - Research Article
Relative Quantification of Proteins in Formalin-Fixed Paraffin-Embedded Breast Cancer Tissue Using Multiplexed Mass Spectrometry Assays
In Brief The identification of biomarkers is an important challenge in oncology. In this study, we developed a liquid chromatography coupled to multiple reaction monitoring mass spectrometry assay that allows measuring the expression of target proteins in formalin-fixed paraffin-embedded tumor samples, the standard method for biopsy storage in clinical pathology. The method provided quantitative information on 185 proteins from three groups of breast tumors: triple-negative, HER2-overexpressing, and luminal A. In addition to markers such as HER2 or hormone receptors, we identified several proteins which are expressed differentially in triple-negative breast cancer samples. - Research Article
Quantitative Phosphoproteomics Analysis Uncovers PAK2- and CDK1-Mediated Malignant Signaling Pathways in Clear Cell Renal Cell Carcinoma
In Brief Senturk et al. (2022) identified >16,000 phosphopeptides in clear cell Renal Cell Carcinoma tumors, of which 600 were determined to be differentially regulated between tumor and normal adjacent tissues. Furthermore, several oncogenic pathways were determined to be enriched in the tumors such as RAC1 activation, MAPK and VEGF signaling, EGFR signaling, and cytokine signaling. Moreover, the kinase PAK2 was identified as one of the key drivers of tumor migration and invasion, having prognostic impact on the survival of ccRCC patients. - Research Article
Mass Spectrometry and Machine Learning Reveal Determinants of Client Recognition by Antiamyloid Chaperones
In Brief A wide range of proteins can prevent amyloid formation in vitro and in vivo, but the exact nature of these interactions is unclear. By combining machine learning–based structure prediction of protein complexes with mass spectrometry, we can discern the molecular determinants of specific and nonspecific chaperone–client interactions. - Research Article
Improving Statistical Certainty of Glycosylation Similarity between Influenza A Virus Variants Using Data-Independent Acquisition Mass Spectrometry
In Brief It is necessary to sample glycoprotein glycoforms deeply for biological studies. DDA is limited by missing values that result from limited scan speed and stochastic precursor ion selection. DIA samples all glycopeptide glycoforms, but duty cycle is reduced compared with DDA. DIA outperforms DDA for confident assignment of glycopeptides provided target glycoproteins are relatively pure. - Research Article
Ionizing Radiation Drives Key Regulators of Antigen Presentation and a Global Expansion of the Immunopeptidome
In-Brief It has been known that cancer treatment using radiation therapy activates the immune system. Here, we show that radiation increases a signaling component of the immune system called the major histocompatibility complex on tumor cells. This increase makes the tumor more recognizable to T cells whose roles are to specifically find and kill defective or mutated cells. These findings could give physicians a reason to combine radiation therapy with immunotherapies to use the immune effect of radiation for therapeutic benefits. - Research Article
The in vivo Interaction Landscape of Histones H3.1 and H3.3
In Brief Proximity-dependent (BioID) interactome analysis of the histone H3 variants H3.1 and H3.3 identified 608 interaction partners and expanded the characterized H3 interaction landscape. Among the most significant findings were that the chaperone chromatin assembly factor 1, previously thought to be H3.1-specific, interacts with H3.3 throughout the cell cycle. Preferential binding was identified between H3.1 and components of the mitotic machinery and between H3.3 and transcription factors, notably MYC interactors. - Research Article
Comparative Proteomic Analysis Identifies Key Metabolic Regulators of Gemcitabine Resistance in Pancreatic Cancer
In Brief For pancreatic cancer (PDAC) patients, gemcitabine resistance (GemR) represents a major clinical problem. Global quantitative differential proteomic analysis of highly GemR PDAC cells developed from relatively Gem-sensitive PDAC cell lines identified multiple networks within the thousands of quantified proteins that support adaptation to Gem-induced stress, and reveal the complexity of GemR. Drug metabolism pathways were key contributors to GemR, and expression changes in Gem-metabolizing enzymes suggest both their influence upon Gem responsiveness, and drug-targetable vulnerabilities to improve clinical Gem efficacy. - Research Article
High-Throughput Human Complement C3 N-Glycoprofiling Identifies Markers of Early Onset Type 1 Diabetes Mellitus in Children
In Brief Changes in glycosylation are known to modulate inflammatory responses, so glycans are more and more being considered as biomarkers of various diseases. Little is known about complement component C3 N-glycome as proper methods for its analysis are lacking, so we developed a novel high-throughput LC-MS method for C3 plasma enrichment and subsequent glycome analysis. We demonstrated that C3 N-glycan profile is stable in a healthy individual, and results showed that C3 N-glycosylation profile is significantly changed in type 1 diabetes. - Research Article
Mass Spectrometry Identification of Biomarkers in Extracellular Vesicles From Plasmodium vivax Liver Hypnozoite Infections
In Brief Human malaria caused by Plasmodium vivax represents a major barrier to achieve the goal of malaria elimination as this species evolved a dormant liver form called a hypnozoite that can remain latent after the initial infection. Current diagnostic tools are unable to detect asymptomatic patients harboring hypnozoites. We have used a MS proteomics approach to discover biomarkers of latent liver infections associated with circulating extracellular vesicles in an in vivo model of P. vivax liver hypnozoite infections. - Research Article
Functional Diversity and Evolution of the Drosophila Sperm Proteome
In Brief Fertilization is the sine qua non of animal and plant reproduction, a process conserved across the tree of life. During fertilization, a specific subset of male-derived proteins, many of unknown function, enter and are present in the developing egg and zygote. We therefore reinterrogated previous published sperm proteomes using improved purification and fractionation methods and identified >3000 proteins in sperm including a novel exchange of paralogous ribosomal proteins suggesting complex patterns of paralog switching and selectivity during spermatogenesis. - Research Article
Temporal and Sex-Linked Protein Expression Dynamics in a Familial Model of Alzheimer’s Disease
In Brief Alzheimer’s disease still lacks effective therapies. In this study, we provide a longitudinal proteomic characterization of 5XFAD mice hippocampus hoping to identify new clinical intervention targets. Our proteomics approach reveals a striking increase in protein expression in 5XFAD mice overtime, which includes 23 proteins here newly associated with Alzheimer’s disease. Globally, our data reveals initial temporal signatures of DNA damage and senescence transitioning into neuroinflammatory signatures concurrent with disease progression. - Research Article
Spatial Proteomics Reveals Differences in the Cellular Architecture of Antibody-Producing CHO and Plasma Cell–Derived Cells
In Brief We used state-of-the-art protein spatial profiling together with MS-based protein expression analysis and subsequent bioinformatics analysis to elucidate the subcellular proteome organization and organelle architecture of two antibody-secreting cell lines, a CHO and a murine plasma derived cell line (MPC-11). Characterization of differentially expressed and localized proteins and their associated biological pathways allowed a systematic comparison of both cell lines and implicates further targets for CHO cell line engineering. - Research Article
Quantitative Acetylomics Reveals Dynamics of Protein Lysine Acetylation in Mouse Livers During Aging and Upon the Treatment of Nicotinamide Mononucleotide
In Brief Li et al. have performed label-free quantification of the acetylomes in mouse livers with age and upon the treatment of nicotinamide mononucleotide (NMN), revealing previously unknown acetylation events. The datasets characterize not just the dynamics of acetylation with age but also specific acetylation sites as new aging biomarkers. The investigation indicates that the life-beneficial effect of NMN could be partially reflected by the changes in age-related protein acetylation. NMN has mild effects on acetylation previously reported as substrates of Sirtuins. - Research Article
Early Prediction of COVID-19 Patient Survival by Targeted Plasma Multi-Omics and Machine Learning
In Brief During times of hospital admission overload, triage may be required to maximize the number of survivors. Mass spectometry–based proteomic and metabolomic analysis of COVID patients’ blood, collected at the time of admission to the ICU, enabled a prediction of survival versus nonsurvival with 92% accuracy. These analyses, which can be performed on widely available mass spectrometers, have the potential to assist physicians with these difficult decisions. - Research Article
Cardiac Myosin Filaments are Maintained by Stochastic Protein Replacement
In Brief To examine the mechanisms of myosin filament replacement in mouse hearts, we labeled proteins with D3-leucine and a GFP-tag in vivo. We used a combination of high-resolution mass spectrometry, analytical modeling, biochemical fractionations, and fluorescence imaging to demonstrate that individual molecules are stochastically replaced from within these large macromolecular filamentous complexes. - Research Article
Quantitative Proteomics Identifies Proteins Enriched in Large and Small Extracellular Vesicles
In Brief The proteome of large and small extracellular vesicles has been determined with quantitative mass spectrometry. Tetraspanins, ADAMs, and ESCRT proteins, as well as SNAREs and Rab proteins associated with endosomes were enriched in small EVs, whereas ribosomal, mitochondrial, and nuclear proteins, as well as proteins involved in cytokinesis, were enriched in large EVs. Several proteins previously suggested to be enriched in either small or large EVs were validated, and several additional novel protein markers were suggested. - Research Article
Phosphoproteomic Analysis of FLCN Inactivation Highlights Differential Kinase Pathways and Regulatory TFEB Phosphoserines
In Brief Comprehensive phosphoproteomics (pY + pSTY) of FLCNPOS and FLCNNEG human renal cells in conjunction with INKA and with posttranslational modification–based signature enrichment analyses identified FLCN phosphorylation dependencies. FLCN-dependent kinase pathways were investigated through drug experiments and validated in a BHD tumor cell line. This pinpointed RTK-MAPK1/3-RPS6K1/3 as a key axis downstream of FLCN loss. In addition, we show that FLCN loss induces ROS and modulates localization of the TFEB transcription factor by dephosphorylation of specific serines. - Research Article
Normics: Proteomic Normalization by Variance and Data-Inherent Correlation Structure
In Brief Normalization of proteomic data is necessary for quantitative comparison and to improve statistical power. Share, extent, and direction of differential expression are usually unknown. Normalizing with unbalanced or high shares of differential expression can distort the data. Normics computes a ranking list for the selection of a likely invariant protein subset for normalization. It increases sensitivity, specificity, and quantitative accuracy compared to standard normalization alone. Its reversed ranking list provides a filter for highly variant proteins for downstream bioinformatic analyses. - Research Article
A Proteomic Approach Identifies Isoform-Specific and Nucleotide-Dependent RAS Interactions
In Brief Miller et al. presents a comprehensive and quantitative interactome of RAS, a protein found to be a driver of many human cancers. This resource identifies interactors of the active form of RAS (nucleotide-dependent) as well as isoform-specific (KRAS, HRAS, and NRAS) interactors of RAS. Several of the proteins identified were confirmed as being important for cancer cell viability or senescence. Exploring these proteins for further studies may lead to new strategies to treat cancers caused by RAS mutations. - Research Article
MS2Rescore: Data-Driven Rescoring Dramatically Boosts Immunopeptide Identification Rates
In Brief The integration of newly trained immunopeptide MS2PIP models, DeepLC, and Percolator into one software package called MS2Rescore allows for a significant boost in immunopeptide identification rate as well as a substantial increase in specificity. MS2Rescore is search engine-agnostic and unbiased toward HLA types. MS2Rescore, therefore, shows great promise to extend the current neo- and xeno-epitope landscape in existing and future immunopeptidomics experiments. - Research Article
Single-Cell Quantitative Proteomic Analysis of Human Oocyte Maturation Revealed High Heterogeneity in In Vitro–Matured Oocytes
In Brief Here, we performed single-cell quantitative proteomic analysis of human germinal vesicle (GV), in vivo (IVO), and in vitro matured (IVM) oocytes and found low correlation between protein and mRNA levels. IVM oocytes showed higher heterogeneity in protein expression, which is related to the levels of estradiol per mature follicle on trigger day. This study provides a rich resource to characterize the mechanisms of oocyte maturation and to evaluate the quality heterogeneity of IVM oocytes at protein level. - Research Article
Limited Evidence for Protein Products of Noncoding Transcripts in the HEK293T Cellular Cytosol
In Brief The discrepancy between the number of novel proteins predicted from ribosomal profiling and detected novel protein products by proteomics remains a matter of debate. We here applied an N-terminal proteogenomics strategy on a HEK293T cytosolic extract to find translational evidence for novel proteins. Our stringent downstream filtering and curation workflow provides limited evidence for the existence of novel proteins and highlights the importance of curation of proteomics data to confidently report novel proteins. - Research Article
Effect of Environmental Temperatures on Proteome Composition of Salmonella enterica Serovar Typhimurium
In Brief We performed systematic proteome analyses of Salmonella enterica serovar Typhimurium (STM) grown at human host body temperature (37 °C) compared to environmental temperatures relevant of agriculture (20, 16, 12, 8 °C). Altered abundance of members of various gene ontology classes reveal global adaptation of environmental temperatures. Virulence proteins for interaction with mammalian hosts were increased at 37 °C, while metabolic proteins were increased at lower temperatures. During growth at 12 °C, STM build functional flagella and showed altered motility. - Research Article
Multiomic Analysis Reveals Disruption of Cholesterol Homeostasis by Cannabidiol in Human Cell Lines
In Brief The nonpsychoactive cannabinoid, cannabidiol (CBD) has been Food and Drug Administration approved for treatment of two drug-resistant epileptic disorders and is seeing widespread supplemental use by the public. The mechanisms that underly the therapeutic effects of CBD are not well understood. We took a systematic multiomics approach to profile the intracellular pathway changes that occur following CBD treatment. From this effort, we identified a response centered in the disruption of cholesterol homeostasis and membrane fluidity that could impact clinical outcomes. - Research Article
Comparative Proteome and Cis-Regulatory Element Analysis Reveals Specific Molecular Pathways Conserved in Dog and Human Brains
In Brief We generated the first spatiotemporal brain proteomes of domestic dogs. By comparison of brain proteomes together with genome-wide cis-regulatory elements frequency between dog, human, and mouse, we identified specific pathways involved in myelination, hippocampus, and neurological diseases conserved between dog and human; the conserved pathways between the two species may help understand their shared social cognitive abilities. The dog brain proteome we generated provides an invaluable resource for comparative studies of brain development, function, and disorder. - Research Article
A Single-Cell Atlas of Tumor-Infiltrating Immune Cells in Pancreatic Ductal Adenocarcinoma
In Brief Here, we employed mass cytometry to characterize the immune composition of PDAC microenvironment. We identified cell clusters with aberrant frequencies in tumors and observed elevated levels of immune markers, for example, PD-1, CD137L, and CD69. Moreover, TCGA PDAC tumors were stratified into three immune subtypes with different prognosis and distinct mechanisms of immune exclusion. Taken together, the data depicted a cell atlas of PDAC immune microenvironment and revealed useful cell phenotypes and targets for immunotherapy development. - Research Article
Larval Exposure to Parasitic Varroa destructor Mites Triggers Specific Immune Responses in Different Honey Bee Castes and Species
In Brief We used three honey bee genotypes and investigated their attractiveness and physiological responses to parasitic Varroa destructor mites. Mites preferred 6-day-old larvae, irrespective of their species/castes. Comparing larval hemolymph proteomes revealed physiological responses to mite exposure in all genotypes and castes, but tolerant larvae responded more pronounced by increasing the abundance of proteins linked to immune and stress responses. We provide key insights into the complex involvement of the honey bee immune system against mite infestations, which could be used for future breeding purposes. - Research Article
In-depth Profiling and Quantification of the Lysine Acetylome in Hepatocellular Carcinoma with a Trapped Ion Mobility Mass Spectrometer
In Brief The proteome and K-acetylome in eight pairs of HCC tumors and normal adjacent tissues was investigated using a timsTOF Pro instrument. We observed suppression of K-acetylation in HCC especially in metabolic enzymes. The roles of deacetylase SIRT2 were explored by examining the effects of SIRT2 overexpression in HCC cells. SIRT2 overexpression reduced K-acetylation on proteins involved in diverse cellular processes and inhibited glycolysis and oxidative phosphorylation. Our findings provide valuable information to understand the roles of K-acetylation in HCC. - Research Article
MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics
In Brief BioID uses a promiscuous biotin ligase to detect protein–protein interactions in a highly reproducible manner. Issues of size, stability, and background labeling of these constructs persist. Here, we modified the structure of BioID2 to create MicroID2, the smallest biotin ligase described. MicroID2 has comparable labeling to TurboID and outperforms miniTurbo. We also developed lbMicroID2, a mutant with lower labeling and less background. Finally, we demonstrated the utility of MicroID2 in mass spectrometry by measuring examining proteins in subcellular organelles. - Research Article
MRG Proteins Are Shared by Multiple Protein Complexes With Distinct Functions
In Brief MRG proteins have been reported to interact with several different factors involved in chromatin modifications, gene regulation, alternative mRNA splicing, and DNA repair. Using genome editing, we tagged endogenous protein to clearly identify stable protein complexes containing these factors. Doing so, we uncovered a new conserved tetrameric complex, named TINTIN, that regulates transcription with reader domains for epigenetic marks. - Research Article
Identification and Analysis of Six Phosphorylation Sites Within the Xenopus laevis Linker Histone H1.0 C-Terminal Domain Indicate Distinct Effects on Nucleosome Structure
In Brief Linker histones are key components of chromosomes and phosphorylated in association with mitosis and other nuclear processes. However, the location of phosphorylation within the high-charged H1 C-terminal domain has been difficult to identify because of its repetitive and highly basic sequence. Moreover, the mechanism by which this post-translational modification alters nucleosome function remains unknown. We identify six sites of modification in the H1 C-terminal domain and demonstrate effects on H1 structure and H1-dependent nucleosome conformation. - Research Article
Differential Proteome and Interactome Analysis Reveal the Basis of Pleiotropy Associated With the Histidine Methyltransferase Hpm1p
In Brief The yeast histidine methyltransferase Hpm1p targets a ribosomal substrate but has many unexplained extraribosomal phenotypes. To understand these, we used protein expression analysis and quantitative cross-linking mass spectrometry to compare WT and Δhpm1 cells. This revealed a role of Hpm1p in metabolism, mitochondria, and membranes. Cross-linking mass spectrometry detected changes in protein structures and interactions and was a powerful means of understanding phenotype. Insights were different to those gained from expression analysis, making these techniques of great potential. - Research Article
The Lysine Acetylation Modification in the Porin Aha1 of Aeromonas hydrophila Regulates the Uptake of Multidrug Antibiotics
In Brief Bacterial antibiotic resistance has become a serious public problem in the world, whereas the intrinsic antibiotic resistance mechanisms are still largely elusive. Protein lysine acetylation (Kac) modification is well distributed in prokaryotic cells and plays important roles in diverse physiological functions. Here, we found that the Kac modifications in a porin family protein Aha1 of Aeromonas hydrophila regulate the uptake of multidrug antibiotics, which may provide a clue for the development of antibiotic therapy strategies. - Research Article
Discovery Proteomics Analysis Determines That Driver Oncogenes Suppress Antiviral Defense Pathways Through Reduction in Interferon-β Autocrine Stimulation
In Brief In this work, we systematically evaluated proteome remodeling driven by well-known oncogenes (c-Myc, EGFR, HER2, AKT, KRAS, BRAF, or MEK) expressed in isogenic models and relevant cancer-derived and patient-derived pancreatic cancer and osteosarcoma. We discovered that these driver oncogenes ubiquitously and dramatically suppressed proteins involved in interferon and antiviral defense pathways. Comprehensive functional and molecular analyses showed that cells expressing oncogenes have reduced interferon autocrine stimulation, impaired dsRNA sensing, and increased susceptibility to infection with an RNA virus. Our results reinforce the direct role of driver oncogenes in establishing an immune evasion phenotype and have immediate relevance to the efficacies of therapies that utilize interferon and antiviral pathways. - Research Article
ImShot: An Open-Source Software for Probabilistic Identification of Proteins In Situ and Visualization of Proteomics Data
In Brief ImShot is the first systematic software that integrates data from IMS and shotgun proteomics (LC–MS) to identify proteins in situ. The software is designed to identify proteins that are particularly responsible for establishing a diseased state. The software usage is independent of any mass spectrometry platform used to generate the data and can also be used for independent proteomics data analysis. ImShot is provided as a desktop application, thereby making its run free of external influencing factors. - Research Article
Interval-Based Secretomics Unravels Acute-Phase Response in Hepatocyte Model Systems
In Brief Using short serum-free incubation intervals allows extended time course analysis downstream of perturbations and addresses limitations of classical serum-free secretomics workflows. The time-dependent secretion of liver cell models HepG2 and HepaRG upon stimulation of the acute-phase response with the proinflammatory cytokines IL1b and IL6 was studied and revealed time- and stimulus-dependent phases of protein secretion. Monitoring proteolysis at the cell surface linked ADAM inhibition to inflammatory cytokine secretion. - Research Article
eEF2K Activity Determines Synergy to Cotreatment of Cancer Cells With PI3K and MEK Inhibitors
In Brief Using a phosphoproteomic approach, we found that eEF2K mediates synergy to treatment with trametinib plus pictilisib (MEK and PI3K inhibitors, respectively). An eEF2K activity signature was highly associated with sensitivity to cotreatment in a panel of 12 AML cell lines. Thus, our study uncovers eEF2K as a key mediator of responses to PI3Ki plus MEKi and a protein activity signature that predicts synergistic response to cotreatments with MEK plus PI3K inhibitors. - Research Article
High Diversity of Glycosphingolipid Glycans of Colorectal Cancer Cell Lines Reflects the Cellular Differentiation Phenotype
In Brief We performed an in-depth analysis of the GSL glycan profiles of 22 CRC cell lines revealing high expression of (sialyl)-LewisA/X and LewisB/Y antigens for colon-like cell lines while undifferentiated cell lines showed higher abundances of glycans with blood group A, B, and H antigens. Combining glycomics with transcriptomic data of relevant genes, significant correlations of Lewis structures with glycosyltransferase FUT3 and transcription factors were revealed, including CDX1. - 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
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
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
Quantitative phosphoproteomics reveals ectopic ATP synthase on mesenchymal stem cells to promote tumor progression via ERK/c-Fos pathway activation
In Brief Accumulating studies demonstrate that mesenchymal stem cells (MSCs) within the tumor microenvironment (TME) play crucial roles in cancer development. The intercellular modulating network between MSCs and lung cancer cells is still unclear. Here, quantitative phosphoproteomics is performed to uncover the phosphosignaling pathway regulated by MSC paracrine factors. Our results suggest that ecto-ATP synthase on the cell surface of MSCs generates eATP into the TME. This eATP enhanced proliferation and migration in lung cancer by activating P2X7R/ERK/phospho-c-Fos-S374 pathway. - 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
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
New Global Insights on the Regulation of the Biphasic Life Cycle and Virulence Via ClpP-Dependent Proteolysis in Legionella pneumophila
In Brief In this work, we present a comprehensive proteomic profile on the life cycle–dependent proteins that are regulated by ClpP-dependent proteolysis and report the temporal regulation of effector expression via the ClpP proteolytic pathway. This study advances our understanding of Legionella in response to different conditions for replication and survival and provides additional evidence that the completion of the biphasic life cycle and bacterial pathogenesis is greatly dependent on protein homeostasis mediated by ClpP-dependent proteolysis. - Research Article
Widespread Arginine Phosphorylation in Staphylococcus aureus
In Brief An optimized workflow for phosphopeptide enrichment revealed extensive protein arginine phosphorylation in S. aureus, which was shown to be stable under the enrichment conditions used. In S. aureus pArg phosphorylation is as widespread as pThr phosphorylation. The phosphatase Stp1 influences the arginine phosphoproteome in an indirect manner, increasing the number of proteins being pArg phosphorylated. It was shown that Stp1 is not a pArg phosphatase itself. - Research Article
Quantitative Proteomic Analysis Reveals Important Roles of the Acetylation of ER-Resident Molecular Chaperones for Conidiation in Fusarium oxysporum
In Brief The present work assessed the importance and levels of acetylation modification in conidial formation of Fusarium oxysporum, demonstrating a critical regulatory role for acetylation in protein biosynthesis and folding during the sporulation process. Acetylation was shown to be modulating the biochemical activities of three endoplasmic reticulum–resident molecular chaperones in F. oxysporum, resulting in a lower level of protein aggregation, which is beneficial for dramatic cell biological remodeling during conidiation. - Research Article
Characterization of the Secretome, Transcriptome, and Proteome of Human β Cell Line EndoC-βH1
In Brief Our knowledge of β cell secretome originates predominantly from rodent studies. Recently, the translatability of rodent β cell secretory profiles has been questioned, emphasizing the need to characterize human β cell secretome in an unbiased manner. Here, we verify that EndoC-βH1 cell line is a representative human β cell model based on RNA-seq and cell lysate proteomics and present the first global secretome proteomics of EndoC-βH1 cells. - Research Article
Imaging Mass Spectrometry Reveals Alterations in N-Linked Glycosylation That Are Associated With Histopathological Changes in Nonalcoholic Steatohepatitis in Mouse and Human
In Brief The rate of nonalcoholic steatohepatitis (NASH) diagnosis has significantly increased, consequently becoming the leading cause of liver transplant in the United States. Modifications to the N-glycome in serum during NASH are of interest to develop clinically relevant strategies for detection. Using MALDI-IMS, we elucidate specific N-glycan alterations that are associated with liver damage histology in NASH. Our results suggest that N-linked fucosylation should be further explored for serum biomarkers development based on the level of fibrosis. - Research Article
Comparative Network Biology Discovers Protein Complexes That Underline Cellular Differentiation in Anabaena sp.
In Brief We reported highly confident protein pairs in vegetative cells and heterocysts of Anabaena sp. PCC 7120, which is the first time such a large protein interaction dataset of Anabaena was generated. Comparing the protein network of the two types of cells can expand our understanding of cell differentiation. Meanwhile, protein–protein interaction data reveal new functions of proteins. We found that the hypothetical protein Alr4359 interacted with FraH and Alr4119 in heterocysts and influenced the diazotrophic growth of filaments. - Research Article
TAILS Identifies Candidate Substrates and Biomarkers of ADAMTS7, a Therapeutic Protease Target in Coronary Artery Disease
In Brief Catalytic inhibition of the secreted enzyme ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) has been proposed as a therapeutic target for coronary artery disease. However, the substrates of ADAMTS7 are not well understood, impeding the understanding of disease biology and preventing the development of activity-based clinical biomarkers. In this study, we provide three discovery sets identifying new ADAMTS7 substrates. We report hundreds of new substrate cleavage sites, including 24 unique cleavage sites found in all ADAMTS7 TAILS experiments. - Research Article
Phosphoproteome Analysis Identifies a Synaptotagmin-1-Associated Complex Involved in Ischemic Neuron Injury
In Brief To investigate the role of protein modification in the hippocampus after cerebral ischemia onset, we used a quantitative proteomics approach to study proteome and phosphoproteome profiles of the acute cerebral ischemic hippocampus in middle cerebral artery occlusion mouse model. Bioinformatic analysis revealed that phosphoproteins fall into a variety of biological processes of synaptic components and neurotransmission. Moreover, functional analysis identifies a synaptotagmin-1-associated complex in mediating ischemic neuron injury. - 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. - Research Article
Phosphoproteome Profiling of the Receptor Tyrosine Kinase MuSK Identifies Tyrosine Phosphorylation of Rab GTPases
In Brief We elucidated phosphosignaling network downstream of muscle-specific receptor tyrosine kinase (MuSK) activated by natural agonist agrin or agonist antibody. Dose–response and time-course experiments showed that both agonists elicited similar intracellular responses. We also characterized two small-molecule inhibitors of MuSK that effectively disrupted MuSK signaling pathway. We further studied the functions of MuSK-responsive tyrosine phosphorylation sites localized on several Rab GTPases. We showed that Rab10 Y6F mutation disrupts association with its adaptor molecules Micals. - 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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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.
