Research Articles
- Research Article
Proteomic Analysis Uncovers Measles Virus Protein C Interaction With p65–iASPP Protein Complex
In Brief Control of measles virus infection and measles-based oncolytic therapy are possible, thanks to the existence of a safe and efficient live attenuated vaccine. Molecular mechanisms that make this vaccine to be so efficient are yet to be determined. We show that the measles C protein is responsible for the establishment of complex networks of interactions with the host cell. We suggest that the C protein binding to the p65–iASPP protein complex controls the host cell death and innate immunity pathways. - Research Article
Dynamic Changes to the Skeletal Muscle Proteome and Ubiquitinome Induced by the E3 Ligase, ASB2β
In Brief The E3 ubiquitin ligase ASB2β has been identified as a regulator of skeletal muscle mass. To gain insights into potential mechanisms of action, mouse muscles expressing a Flag-tagged ASB2β were investigated using quantitative proteomic methods. The results identified ASB2β-induced changes in the abundance and ubiquitination of proteins associated with mitochondria, the sarcomere, and the cytoskeleton. Additional in vitro studies identified novel putative ASB2β target substrates. The results highlight the complex relationship between protein abundance and ubiquitination in ASB2β-mediated muscle adaptation. - Research Article
New Proteomic Signatures to Distinguish Between Zika and Dengue Infections
In Brief Differentiation between the mosquito-borne Zika and dengue Flavivirus infections is clinically important for correct treatment, but remains challenging. We used mass spectrometry to quantify expression levels for 277 proteins measured in serum samples from 62 patients, providing a resource to the community. We identified 13 proteins with significant differential expression between the closely related types of infections. Most of the proteins link to pregnancy and brain function. We also identified expression signatures that mark ambiguous infections with respect to temporal differences. - Research Article
Protein Phosphorylation in Depolarized Synaptosomes: Dissecting Primary Effects of Calcium from Synaptic Vesicle Cycling
In Brief Analysis of protein phosphorylation in isolated nerve terminals (synaptosomes) treated with C. botulinum neurotoxins (BoNT) to inhibit synaptic vesicle (SV) cycling reveals phosphorylation events that are primarily dependent on depolarization-induced Ca2+ influx and those that also require active SV-cycling machinery. In particular, SV-cycling-dependent phosphorylation sites on synaptobrevin (Vamp2), syntaxin-1 (Stx1a), and cannabinoid receptor-1 (Cnr1) are capable of changing the rate of exo- and endocytosis in cultured hippocampal neurons. - Research Article
Quantitative Proteomics and Phosphoproteomics Support a Role for Mut9-Like Kinases in Multiple Metabolic and Signaling Pathways in Arabidopsis
In Brief The MUT9-like kinases are a family of plant-specific nuclear-localized kinases with roles in diverse signaling pathways, including light sensing, phytohormone perception, and the circadian clock. The proteome and phosphoproteome of compound mlk mutant seedlings have been determined under light and dark conditions. These experiments identify new roles for these kinases regulating secondary plant metabolism and stress responses, tested through metabolite analysis and assaying seedling sensitivity to DNA damaging agents. - Research Article
Proximity-dependent Mapping of the Androgen Receptor Identifies Kruppel-like Factor 4 as a Functional Partner
In Brief A proximity interaction network for the androgen receptor (AR) was obtained from androgen-responsive prostate cancer cells. A total of 267 candidates were identified, most associating following ligand stimulation, including Krüppel-like factor 4 (KLF4). KLF4 and AR were found to colocalize genome-wide on 4097 genes including PSA (KLK3), for which KLF4 acts as a repressor, without regulating the expression of AR. These results are instrumental to further dissect the molecular mechanisms underlying androgen signaling in prostate cells. - Technological Innovation and Resources
Stable Isotope Labeling of Amino Acids in Flies (SILAF) Reveals Differential Phosphorylation of Mitochondrial Proteins Upon Loss of OXPHOS Subunits
In Brief We advanced a fully composed food source for Drosophila melanogaster into a highly efficient, versatile, and cheap labeling method, termed SILAF. The larval proteome incorporates more than 99% heavy lysine-6 label within 6 days, while the adult fly metabolism allows protein turnover studies. We obtained a phosphoproteome with site-specific occupancy in a fly model of mitochondrial metabolic disease, highlighting the regulation of two novel conserved phosphosites on subunits of the electron transport chain. - Research Article
Secretome and Comparative Proteomics of Yersinia pestis Identify Two Novel E3 Ubiquitin Ligases That Contribute to Plague Virulence
In Brief Rapid adaption to the environments is critical for microbes to establish infection. Quantitative proteome and secretome analyses of Y. pestis grown under conditions mimicking its two typical natural niches were performed to understand the adaption strategies of this deadly pathogen. We identified three secreted proteins that can be translocated into host cells and further demonstrated that two of them show strong E3 ubiquitin ligase activity and contribute significantly to the virulence of Y. pestis. - Research Article
Proteomic Profiling of Gastric Signet Ring Cell Carcinoma Tissues Reveals Characteristic Changes of the Complement Cascade Pathway
In Brief This study took advantages of LCM and DIA-MS, generating a data set in the context of different subtypes of gastric tumors, globally and precisely. It was discovered for the first time that the complement cascade in SRCC tumors was specifically activated compared with AC. - Research Article
Quantitative Proteomics Reveals that the OGT Interactome Is Remodeled in Response to Oxidative Stress
In Brief The goal of these studies was to provide insight into the regulation of the O-GlcNAc transferase (OGT) basally and in response to oxidative stress, as well as the role that O-GlcNAc plays in promoting cytoprotection. Using quantitative proteomics, the basal and injury-induced interactome of OGT has been defined and validated. Protein interactors are anticipated to regulate either the activity or substrate targeting of OGT, or to be substrates of OGT, thus affecting cytoprotection. - Research Article
Integrative Proteomic and Metabolomic Analysis Reveals Metabolic Phenotype in Mice With Cardiac-Specific Deletion of Natriuretic Peptide Receptor A
In Brief Metabolomic analysis in mice revealed that natriuretic peptide receptor A (NPRA) is mainly involved in nucleotide biosynthesis and histidine metabolism in cardiac tissues, and in creatine metabolism, TCA cycle and pentose phosphate pathway in the plasma. Furthermore, proteomics revealed that Cox7c, Cox7b, ATP5J2, Uqcr10, and Myh7 play a vital role in the regulation of metabolic pathway. Together, deterioration of NPRA results in metabolic dysfunction involved with a protein and metabolite-interacting pathway. - Research Article
Integrated Redox Proteomic Analysis Highlights New Mechanisms of Sensitivity to Silver Nanoparticles
In Brief Silver nanoparticles (AgNPs) are widely used nanomaterials, but the molecular mechanisms underlying their activity remain elusive. Here, we show using time course studies that AgNP-sensitive lung cells experience broader and more pronounced changes in protein abundance and protein oxidation impacting protein translation and modification, lipid metabolism, bioenergetics, and mitochondrial dynamics. The findings are further validated by imaging of mitochondria using confocal microscopy and transmission electron microscopy. - Research Article
Domain Mapping of Chondroitin/Dermatan Sulfate Glycosaminoglycans Enables Structural Characterization of Proteoglycans
In Brief Glycosaminoglycans (GAGs) remain one of the most challenging posttranslational modifications to study, much due to their structural complexity and heterogeneity, and new methods for analysis are therefore required. We have developed a protocol for enrichment and structural characterization of GAGs of proteoglycans using nLC-MS/MS. We provide detailed information on the nonreducing end, internal, and linkage region GAG domains and use the data to determine an overall GAG structure of chromogranin-A of rat INS-1832/13 cells. - Research Article
Mapping Isoform Abundance and Interactome of the Endogenous TMPRSS2-ERG Fusion Protein by Orthogonal Immunoprecipitation–Mass Spectrometry Assays
In Brief Orthogonal immunoprecipitation-mass spectrometry assays quantified TMPRSS2-ERG fusion protein (∼27,000 copies/cell) and its four distinct isoforms, and revealed that T1E4-ERG isoform accounted for 52 ± 3% of the total ERG in VCaP cells and 50 ± 11% in FFPE prostate cancer tissues. Methionine-truncated and N-acetylated peptide TASSSSDYGQTSK unique for T1/E4 TMPRSS2-ERG fusion was identified. Unlike the N-terminal antibodies, C-terminal antibodies identified 29 ERG-interacting proteins, including mutually exclusive BRG1- and BRM-associated canonical SWI/SNF chromatin remodeling complexes. Clinical perspectives of assays were discussed. - Research Article
In-depth Site-specific Analysis of N-glycoproteome in Human Cerebrospinal Fluid and Glycosylation Landscape Changes in Alzheimer's Disease
In Brief An exploratory glycosylation-based biomarker study has been conducted for in-depth mapping of an overall glycosylation landscape and site-specific alteration in glycoproteome collected from cerebrospinal fluids (CSF) in healthy control and Alzheimer’s disease (AD) subjects. The comparison will shed light on the glycoproteome profile, dominant glycosylation differences and similarities, and some of the interesting glycoprotein candidates with specific glycosylation pattern alterations in AD. - Research Article
Feature Selection Methods for Protein Biomarker Discovery from Proteomics or Multiomics Data
In Brief Untargeted mass spectrometry–based proteomics provides a powerful platform for protein biomarker discovery, but clinical translation depends on the selection of a small number of proteins for verification and validation. We present feature selection methods for protein biomarker selection from proteomics or multiomics data. The algorithms show good performance, enable functional interpretation of the identified markers, and provide alternative choices for each identified marker to facilitate a robust transition to the verification and validation platforms. - Technological Innovation and Resources
Enabling Photoactivated Cross-Linking Mass Spectrometric Analysis of Protein Complexes by Novel MS-Cleavable Cross-Linkers
In Brief Although photochemistry complements residue-specific chemistry through labeling amino acids nonspecifically, existing photo-cross-linking reagents are thus far inapplicable to multisubunit protein complexes owing to low yields and high complexities of photo-cross-linked products. The development of the three sulfoxide-containing MS-cleavable photoreactive SDASO cross-linkers permits MSn-based analytical workflow for accurate identification of photo-cross-linked peptides, enabling complex PPI profiling for the first time. This work has established a solid foundation for future applications of photo-cross-linking in complex XL-MS studies. - Research Article
Calculating Sample Size Requirements for Temporal Dynamics in Single-Cell Proteomics
In Brief Cellular development and disease progression are gradual transitions between phenotypic stages. Time-course measurements that explicitly measure this transition are important to discover proteome dynamics. Single-cell measurements are a powerful tool for understanding heterogeneity, especially during phenotypic transitions. Single-cell proteomics measurements are emerging as an available tool to characterize the cellular state. We created a statistical method that predicts the success of an experimental design for temporal dynamics. - Research Article
TMEM67, TMEM237, and Embigin in Complex With Monocarboxylate Transporter MCT1 Are Unique Components of the Photoreceptor Outer Segment Plasma Membrane
In Brief The plasma membrane which envelopes the light-sensitive outer segment organelle of vertebrate photoreceptor cells plays diverse roles in supporting photoreceptor function and health. Protein correlation profiling of this membrane revealed a surprisingly small number of unique protein components. Among them are TMEM67 and TMEM237, whose mutations are associated with various syndromic ciliopathies, and embigin found to be associated with the monocarboxylate transporter MCT1. The MCT1–embigin complex likely facilitates lactate transport through this cellular compartment. - Research Article
HLA Class II Presentation Is Specifically Altered at Elevated Temperatures in the B-Lymphoblastic Cell Line JY
In Brief Here the cellular response in a “fever-mimicking state” was investigated by sampling in parallel the proteome and the HLA class I and II ligandomes. Using quantitative proteomics and immunopeptidomics, we found that a proteomic “fever response” is initiated in B-cells after growing the cells for only 3 days at 40 °C and that it is largely mediated by adaptation in the HLA class II rather than HLA class I system. - Research Article
Structural Investigations of Human A2M Identify a Hollow Native Conformation That Underlies Its Distinctive Protease-Trapping Mechanism
In Brief The native conformation of the protease inhibitor A2M was investigated using negative stain electron microscopy, small-angle X-ray scattering, and cross-linking mass spectrometry. The low-resolution model built from these data describes a hollow tubular configuration that explains several aspects of A2M’s unique trapping mechanism. This model was further validated by two recombinantly expressed A2M mutants, which probed the location of the bait region and demonstrated the existence of a critical interface between A2M’s disulfide-bridged dimers. - Research Article
The Expression of NTAL and Its Protein Interactors Is Associated With Clinical Outcomes in Acute Myeloid Leukemia
In Brief Here, we demonstrated that the knockdown of non–T cell activation linker (NTAL) in acute myeloid leukemia (AML) cells was linked to reduced cell proliferation and survival in vitro and in vivo. In addition, we identified NTAL interactors in AML using label-free protein quantification. NTAL interactors presented a high expression in patients with AML, being associated with a leukemic granulocyte–macrophage progenitor-like state. Finally, NTAL interactors were capable to predict survival in a large subset of patients with AML. These data provide evidence that NTAL and its interactors could represent potential therapeutic targets for granulocyte–macrophage progenitor-like leukemias. - Research Article
The C-Mannosylome of Human Induced Pluripotent Stem Cells Implies a Role for ADAMTS16 C-Mannosylation in Eye Development
In Brief We identified ADAMTS16 as a target protein for C-mannosylation and showed that this modification is needed for proper secretion of ADAMTS16. Targeting a distinct C-mannosyltransferase by CRISPR–Cas9 in medaka fish embryos caused defects in eye development. We conclude that these developmental defects are caused by reduced secretion of ADAMTS16 when C-mannosylation is missing. - Technological Innovation and Resources
GAGrank: Software for Glycosaminoglycan Sequence Ranking Using a Bipartite Graph Model
In Brief We demonstrate GAGrank, an algorithm that uses a bipartite graph model for sequencing glycosaminoglycans from EDD or NETD tandem mass spectra. The process involves first assigning glycosaminoglycan product ions using the GAGfinder algorithm. The second step is to rank possible sequences using GAGrank. We show GAGrank’s ability to sequence isomeric mixtures. - Research Article
Reverse Phase Protein Array Reveals Correlation of Retinoic Acid Metabolism With Cardiomyopathy in Friedreich's Ataxia
In Brief We conducted a sensitive and high-throughput reverse phase protein array to attain protein expression profiles of primary fibroblasts from patients with Friedreich's ataxia (FRDA) and unaffected controls using a pool of 217 validated antibodies. Our extensive bioinformatics analyses correlated differentially expressed (DE) proteins with critical disease parameters. Expression levels of several integrin proteins specifically associated with hearing loss in FRDA. Also, reverse phase protein array data integrated with transcriptome data uncovered defects in retinoic acid metabolism in FRDA samples.
