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.
