Research Articles
- Research Article
In-Cell Chemical Crosslinking Identifies Hotspots for SQSTM-1/p62-IκBα Interaction That Underscore a Critical Role of p62 in Limiting NF-κB Activation Through IκBα Stabilization
In Brief The transcriptional activator NF-κB inhibitor, IκBα is proteolytically unstable when uncomplexed. How newly synthesized IκBα escapes degradation to terminate nuclear NF-κB activation is unknown. In-cell chemical crosslinking and proximity labeling MS analyses uncovered a novel association of p62 with IκBα via well-defined structural hotspots, which impairs its interaction with 26S/20S proteasome, extends its life-span and enables its termination of NF-κB activation. Mice carrying liver-specific genetic deletion of p62-IκBα hotspot exhibit enhanced liver inflammation upon aging, validating this novel p62 role. - Research Article
Lysine Succinylation of VBS Contributes to Sclerotia Development and Aflatoxin Biosynthesis in Aspergillus flavus
In Brief In this research, by comparing the succinylation levels of the two strains and exploring the relationship between succinylation and aflatoxin synthesis, our succinylome data identified 1240 lysine succinylation sites in 768 proteins, among which 1103 lysine succinylation sites in 685 proteins were quantified, the majority of proteins involved in aflatoxin biosynthetic pathway were downregulated in high aflatoxin-producing strains. In function analysis, we confirmed that K135 has a critical effect on VBS activity, maintenance of sclerotia and aflatoxin production. - Research Article
HIV-1 Transcription Inhibitor 1E7-03 Decreases Nucleophosmin Phosphorylation
In Brief Small molecule HIV-1 transcription inhibitor, 1E7-03, that binds to the noncatalytic RVxF accommodating site of protein phosphatase 1 reprogramed protein phosphorylation in the PPARα/RXRα, TGF-β, and PKR pathways. Phosphorylation of nucleophosmin (NPM1) at Ser-125 residue was significantly reduced. NPM1 S125D mutant activated Tat-induced HIV-1 transcription and exhibited enhanced NPM1–HIV-1 Tat interaction. Inhibition of Aurora A or Aurora B kinases that phosphorylate NPM1 inhibited HIV-1. Our findings suggest that NPM1 phosphorylation is a plausible target for HIV-1 transcription inhibition. - Research Article
Native Size-Exclusion Chromatography–Based Mass Spectrometry Reveals New Components of the Early Heat Shock Protein 90 Inhibition Response Among Limited Global Changes
In Brief Samant & Batista et al. use native SEC-MS to characterize how protein complexes are modulated in human cancer cells treated with tanespimycin, a clinically developed inhibitor of the molecular chaperone HSP90. Among 4645 proteins identified across at least three of the four replicates, the authors unexpectedly find only limited changes to global protein distributions, rather than proteome-wide remodeling, following HSP90 inhibition. Nevertheless, several novel HSP90-dependent components are identified through this dataset, including Anillin and mitochondrial IDH3. - Research Article
Oxonium Ion–Guided Optimization of Ion Mobility–Assisted Glycoproteomics on the timsTOF Pro
In Brief In this study, we advance the use of the timsTOF Pro by designing and applying a novel acquisition routine for glycoproteomics. We demonstrate that the instrument can be specifically focused on sequencing glycopeptides by making use of ion mobility, and that its high scan rate can be used to improve spectrum quality. This can be applied to great effect as we show that, especially for shorter gradients, the number of glycopeptide detections can be sustained at a high level. - Research Article
Resistin, Elastase, and Lactoferrin as Potential Plasma Biomarkers of Pediatric Inflammatory Bowel Disease Based on Comprehensive Proteomic Screens
In Brief Inflammatory bowel disease (IBD) is an immune-mediated chronic inflammation of the intestine, which can present as ulcerative colitis or Crohn’s disease. Plasma samples from a pediatric IBD cohort of 22 subjects were interrogated using an aptamer-based screen of 1322 proteins, and the elevated biomarkers identified were further validated by ELISA using an independent cohort of 76 pediatric plasma samples. We have identified circulating resistin, elastase, and lactoferrin as potential plasma biomarkers of IBD in pediatric patients. - Research Article
MSstatsPTM: Statistical Relative Quantification of Posttranslational Modifications in Bottom-Up Mass Spectrometry-Based Proteomics
In Brief MSstatsPTM detects statistical differences between posttranslational modifications and global protein abundances. It includes novel methodology to remove confounding between the effect of individual modification versus the global protein. MSstatsPTM is implemented as an open-source R package available on Bioconductor. The package can be applied to a variety of experimental designs, including those which are label-free and label-based. The package has been benchmarked on several datasets including simulated datasets, a spike-in controlled investigation, and multiple biological experiments. - Research Article
Proteomic Profiling Reveals the Molecular Control of Oocyte Maturation
In Brief Using TMT-based LC-MS/MS, we systematically performed a quantitative proteomic analysis of GV, GVBD, and MII oocytes. We successfully quantified 4694 proteins from 4500 oocytes in three key stages. By combining bioinformatics analysis with functional approaches, our results showed the protein kinetics and molecular mechanisms during meiotic maturation. - Research Article
Mass Spectrometry–Driven Discovery of Neuropeptides Mediating Nictation Behavior of Nematodes
In Brief Neuropeptides are a type of signaling molecules that regulate certain behaviors. One such behavior is nictation, a strategy used by pathogenic nematodes for host finding. These pathogenic nematodes are often commercially significant, as they can be utilized as biopesticides in agriculture. Here, we have developed a method using mass spectrometry to identify and quantify neuropeptides responsible for nictation behavior. As a result, we were able to demonstrate that neuropeptide genes flp-7 and flp-11 are novel regulators of nictation. - Research Article
Proteome Remodeling of the Eye Lens at 50 Years Identified With Data-Independent Acquisition
In Brief The lens microcirculation system is essential for maintenance of lens transparency. To evaluate maintenance of the microcirculatory system, we measured three developmentally distinct fiber cell populations within 16 human lenses of increasing age. Using data-independent acquisition, we identified a proteome remodeling event that occurs at approximately 50 years of age. Age-modified proteins included gap junctions, a calcium transporter, aquaporin-5, and oxidative stress response–related proteins. Remodeling may result in calcium-activated protease activity and disruption of the microcirculation system. - Research Article
The First Pituitary Proteome Landscape From Matched Anterior and Posterior Lobes for a Better Understanding of the Pituitary Gland
In Brief Our study of the first pituitary proteome draft offers the following novelty; for the first time, we present an extensive proteomic database containing proteins from both the anterior and posterior lobes of healthy pituitary, identifying various pituitary-enriched proteins and their lobe specificities, followed by target verification of pituitary hormones; for the first time, we are describing three uPE1 and have demonstrated that S100 proteins, which are known markers for pituitary adenomas, showed their presence significantly in the posterior lobe. - 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.
