Research Articles
- Research Article
Increasing Complexity of the N-Glycome During Caenorhabditis Development
In Brief There is an increasing N-glycomic complexity during development of the nematode, Caenorhabditis elegans, as revealed by off-line HPLC/MALDI-TOF-MS/MS. The higher degree of glycan methylation and α-galactosylation of mannose residues in liquid-grown worms may reflect cultivation-dependent stress. Furthermore, phosphorylcholine modifications were found not just on N-glycans but on O-glycans. The increased branching and core fucosylation of N-glycans in the larvae as compared with the embryos may correlate with regulated expression of key glycosyltransferases. - Research Article
P300 Interacted With N-Myc and Regulated Its Protein Stability via Altering Its Post-Translational Modifications in Neuroblastoma
In Brief Endogenous and exogenous N-Myc coimmunoprecipitation combined with HPLC–MS/MS identified 16 PTM residues of N-Myc and 114 potential N-Myc-interacting proteins. Among them, p300 interacted with N-Myc and modulated the protein stability of N-Myc by simultaneously regulating the acetylation and ubiquitination level on lysine-199 of N-Myc protein in vitro. Furthermore, p300 promoted cell proliferation and invasion of MYCN-amplified NB cells and correlated with poor prognosis in NB patients, making p300 a potential therapeutic target for MYCN-amplified NB patients. - Research Article
Synergistic Targeting of DNA-PK and KIT Signaling Pathways in KIT Mutant Acute Myeloid Leukemia
In Brief This study provides insight into the oncogenic pathways regulated by KIT, revealing an important role for DNA-PK activation, beyond its canonical role in DNA repair. DNA-PK activation may be a common event in receptor tyrosine kinase mutant AML and is a promising novel therapeutic target for this poor-prognosis AML subtype. - Research Article
O-Glycomic and Proteomic Signatures of Spontaneous and Butyrate-Stimulated Colorectal Cancer Cell Line Differentiation
In Brief Bacterial butyrate is beneficial for intestinal homeostasis as the preferred energy source of intestinal epithelia, capable of inducing differentiation. Here, we describe glycomic and proteomic signatures of butyrate-induced epithelial differentiation of the intestinal cell line CaCo-2. We identified an upregulation of specific O-glycan signatures as well as specific proteins involved in cell metabolism, monosaccharide precursor biosynthesis, and cell adhesion. Using an integrative approach, we generated hypotheses about the origin of the glycosylation changes with differentiation. - Research Article
Proteomic Discovery of Plasma Protein Biomarkers and Development of Models Predicting Prognosis of High-Grade Serous Ovarian Carcinoma
In Brief To investigate novel, prognostic protein biomarkers, we conducted label-free liquid chromatography–tandem mass spectrometry using frozen plasma samples obtained from patients with newly diagnosed high-grade serous ovarian carcinoma. Candidate biomarkers underwent validation with an independent set of plasma samples via ELISA. By combining clinical factors and ELISA results, we successfully developed models and nomograms to predict the 18-month progression-free survival rate for clinical use. - Research Article
Capture, Release, and Identification of Newly Synthesized Proteins for Improved Profiling of Functional Translatomes
In Brief In this study, we have optimized the O-propargyl-puromycin (OPP)-mediated identification of nascent proteins by incorporating a cleavable linker, Dde biotin-azide, into our protocol. Following capture on streptavidin agarose resin, nascent polypeptides were selectively released from the resin by treatment with 2% hydrazine. Using the cleavable linker reduced the presence of background proteins and facilitated the detection of low-abundance proteins. We profiled the nascent proteome upon acute inhibition of mTOR and observed downregulation of many proteins involved in translational initiation. - 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.
