Proteomic analysis reveals the negative modulator of sperm function glycodelin as over-represented in semen exosomes isolated from asthenozoospermic patients

2019 ◽  
Vol 34 (8) ◽  
pp. 1416-1427 ◽  
Author(s):  
Valentina Murdica ◽  
Greta Chiara Cermisoni ◽  
Natasa Zarovni ◽  
Andrea Salonia ◽  
Paola Viganò ◽  
...  
Keyword(s):  
Western Blot ◽  
Proteomic Analysis ◽  
Large Scale ◽  
Reproductive Tract ◽  
Secretory Protein ◽  
Differentially Expressed ◽  
Mass Spectrometry Analysis ◽  
Inhibitory Protein ◽  
Reproductive Process ◽  
Spectrometry Analysis

ABSTRACT STUDY QUESTION Are there differences in the proteomic profile of exosomes isolated from seminal plasma of normozoospermic (NSP) and severe asthenozoospermic (SA) men, potentially contributing to sperm features? SUMMARY ANSWER A relevant group of proteins known to positively regulate sperm functions were over-represented in seminal exosomes of NSP men, i.e. cysteine-rich secretory protein-1 (CRISP1), while the inhibitory protein glycodelin was enriched in exosomes of SA subjects. WHAT IS KNOWN ALREADY Exosomes are secreted along the male reproductive tract and are thought to be involved in spermatozoa maturation and function. Ejaculated spermatozoa are still able to capture exosomes; exosomes of NSP individuals improve sperm motility and prompt capacitation, while exosomes of SA men fail to exert similar features. STUDY DESIGN, SIZE, DURATION Semen samples from NSP and SA men, aged 18 to 55 and registered at a single IVF center, were considered for this study project. Subjects were subdivided into three groups: a discovery cohort (five NSP men and six SA patients), a validation cohort (seven NSP and seven SA men) and the ‘glycodelin analysis’ cohort (20 NSP and 37 SA men). Exosomes were purified from semen of every participant. PARTICIPANTS/MATERIALS, SETTING, METHODS Exosomes were characterized by nanoparticle tracking analysis, transmission electron microscopy and western blot. Comprehensive proteomics analysis of the exosomal proteome was performed by nanoscale liquid chromatographic tandem mass spectrometry analysis. Funrich software was used to determine statistical enrichment of pathways, networks and Gene Ontology terms of the identified proteins. Validation of differentially expressed proteins was performed through ELISA and western blot analysis. MAIN RESULTS AND THE ROLE OF CHANCE The comprehensive proteomic analysis identified a total of 2138 proteins for both groups. There were 89 proteins found to be differentially expressed in exosomes of NSP versus SA subjects, of which 37 were increased in the NSP group and 52 were increased in the SA group. One-third of the exosomes-associated proteins highly expressed in NSP samples were involved in the reproductive process; conversely, the over-expressed proteins in exosomes of SA samples were not functionally specific. Quantitative data were confirmed on seminal exosomes from different cohorts of subjects. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION Transfer of the proteins from exosomes to spermatozoa has been only partially demonstrated and up-take mechanisms are still poorly defined. WIDER IMPLICATIONS OF THE FINDINGS Seminal exosomes carry proteins that are potentially able to either favour or inhibit the reproductive process in humans. A better understanding of these phenomena might pave the way for novel intervention measures in terms of male infertility. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by the Italian Ministry of Health through an Institution Seed Grant. None of the authors has any competing interests.

scholarly journals Proteomics Analysis of SsNsd1-Mediated Compound Appressoria Formation in Sclerotinia sclerotiorum

2018 ◽  
Vol 19 (10) ◽  
pp. 2946 ◽  
Author(s):  
Jingtao Li ◽  
Xianghui Zhang ◽  
Le Li ◽  
Jinliang Liu ◽  
Yanhua Zhang ◽  
...  
Keyword(s):  
Sclerotinia Sclerotiorum ◽  
Differentially Expressed ◽  
Mass Spectrometry Analysis ◽  
Differentially Expressed Proteins ◽  
Transcript Accumulation ◽  
Spectrometry Analysis ◽  
Peptide Mass ◽  
Appressoria Formation ◽  
Dimensional Electrophoresis ◽  
Shed Light

Sclerotinia sclerotiorum (Lib.) de Bary is a devastating necrotrophic fungal pathogen attacking a broad range of agricultural crops. In this study, although the transcript accumulation of SsNsd1, a GATA-type IVb transcription factor, was much lower during the vegetative hyphae stage, its mutants completely abolished the development of compound appressoria. To further elucidate how SsNsd1 influenced the appressorium formation, we conducted proteomics-based analysis of the wild-type and ΔSsNsd1 mutant by two-dimensional electrophoresis (2-DE). A total number of 43 differentially expressed proteins (≥3-fold change) were observed. Of them, 77% were downregulated, whereas 14% were upregulated. Four protein spots fully disappeared in the mutants. Further, we evaluated these protein sequences by mass spectrometry analysis of the peptide mass and obtained functionally annotated 40 proteins, among which only 17 proteins (38%) were identified to have known functions including energy production, metabolism, protein fate, stress response, cellular organization, and cell growth and division. However, the remaining 23 proteins (56%) were characterized as hypothetical proteins among which four proteins (17%) were predicted to contain the signal peptides. In conclusion, the differentially expressed proteins identified in this study shed light on the ΔSsNsd1 mutant-mediated appressorium deficiency and can be used in future investigations to better understand the signaling mechanisms of SsNsd1 in S. sclerotiorum.

scholarly journals Proteomic analysis of differentially expressed proteins in normal human thyroid cells transfected with PPFP

2012 ◽  
Vol 19 (5) ◽  
pp. 681-694 ◽  
Author(s):  
Xinying Li ◽  
Zhiming Wang ◽  
Jianming Liu ◽  
Cane Tang ◽  
Chaojun Duan ◽  
...  
Keyword(s):  
Western Blot ◽  
Proteomic Analysis ◽  
Colony Formation ◽  
Fusion Gene ◽  
Differentially Expressed ◽  
Human Thyroid ◽  
Peroxisome Proliferator ◽  
Differentially Expressed Proteins ◽  
Thyroid Cells ◽  
Peroxisome Proliferator Activated Receptor

The fusion gene encoding the thyroid-specific transcription factor PAX8 and peroxisome proliferator-activated receptor γ (PPARγ (PPARG)) (designated as the PPFP gene) is oncogenic and implicated in the development of follicular thyroid carcinoma (FTC). The effects of PPFP transfection on the biological characteristics of Nthy-ori 3-1 cells were studied by MTT assay, colony formation, soft-agar colony formation, and scratch wound-healing assays as well as by flow cytometry. Furthermore, the differentially expressed proteins were analyzed on 2-DE maps and identified by MALDI-TOF-MS. Validation of five identified proteins (prohibitin, galectin-1, cytokeratin 8 (CK8), CK19, and HSP27) was determined by western blot analysis. PPFP not only significantly increased the viability, proliferation, and mobility of the Nthy-ori 3-1 cells but also markedly inhibited cellular apoptosis. Twenty-eight differentially expressed proteins were identified, among which 19 proteins were upregulated and nine proteins were downregulated in Nthy-ori 3-1PPFP(Nthy-ori 3-1 cells transfected with PPFP). The western blot results, which were consistent with the proteome analysis results, showed that prohibitin was downregulated, whereas galectin-1, CK8, CK19, and HSP27 were upregulated in Nthy-ori 3-1PPFP. Our results suggest that PPFP plays an important role in malignant thyroid transformation. Proteomic analysis of the differentially expressed proteins in PPFP-transfected cells provides important information for further study of the carcinogenic mechanism of PPFP in FTCs.

scholarly journals Co-Localization of Crotamine with Internal Membranes and Accentuated Accumulation in Tumor Cells

Molecules ◽  
2018 ◽  
Vol 23 (4) ◽  
pp. 968 ◽  
Author(s):  
Nicole Mambelli-Lisboa ◽  
Juliana Mozer Sciani ◽  
Alvaro Rossan Brandão Prieto da Silva ◽  
Irina Kerkis
Keyword(s):  
Molecular Mass ◽  
Tumor Cells ◽  
Large Scale ◽  
Time Lapse ◽  
Mass Spectrometry Analysis ◽  
Molecular Probe ◽  
Spastic Paralysis ◽  
Spectrometry Analysis ◽  
Hind Limbs ◽  
Wide Range

Crotamine is a highly cationic; cysteine rich, cross-linked, low molecular mass cell penetrating peptide (CPP) from the venom of the South American rattlesnake. Potential application of crotamine in biomedicine may require its large-scale purification. To overcome difficulties related with the purification of natural crotamine (nCrot) we aimed in the present study to synthesize and characterize a crotamine analog (sCrot) as well investigate its CPP activity. Mass spectrometry analysis demonstrates that sCrot and nCrot have equal molecular mass and biological function—the capacity to induce spastic paralysis in the hind limbs in mice. sCrot CPP activity was evaluated in a wide range of tumor and non-tumor cell tests performed at different time points. We demonstrate that sCrot-Cy3 showed distinct co-localization patterns with intracellular membranes inside the tumor and non-tumor cells. Time-lapse microscopy and quantification of sCrot-Cy3 fluorescence signalss in living tumor versus non-tumor cells revealed a significant statistical difference in the fluorescence intensity observed in tumor cells. These data suggest a possible use of sCrot as a molecular probe for tumor cells, as well as, for the selective delivery of anticancer molecules into these tumors.

scholarly journals Identification of cytoplasmic sialidase NEU2-associated proteins by LC-MS/MS

2018 ◽  
Vol 44 (4) ◽  
pp. 462-472
Author(s):  
Secil Akyildiz Demir ◽  
Volkan Seyrantepe
Keyword(s):  
Mass Spectrometry ◽  
Western Blot ◽  
Blot Analysis ◽  
Actin Filaments ◽  
Western Blot Analysis ◽  
Mammalian Cells ◽  
Protein S ◽  
Mass Spectrometry Analysis ◽  
Interacting Protein ◽  
Spectrometry Analysis

Abstract Background Cytoplasmic sialidase (NEU2) plays an active role in removing sialic acids from oligosaccharides, glycopeptides, and gangliosides in mammalian cells. NEU2 is involved in various cellular events, including cancer metabolism, neuronal and myoblast differentiation, proliferation, and hypertrophy. However, NEU2-interacting protein(s) within the cell have not been identified yet. Objective The aim of this study is to investigate NEU2 interacting proteins using two-step affinity purification (TAP) strategy combined with mass spectrometry analysis. Methods In this study, NEU2 gene was cloned into the pCTAP expression vector and transiently transfected to COS-7 cells by using PEI. The most efficient expression time of NEU2- tag protein was determined by real-time PCR and Western blot analysis. NEU2-interacting protein(s) were investigated by using TAP strategy combined with two different mass spectrometry experiment; LC-MS/MS and MALDI TOF/TOF. Results Here, mass spectrometry analysis showed four proteins; α-actin, β-actin, calmodulin and histone H1.2 proteins are associated with NEU2. The interactions between NEU2 and actin filaments were verified by Western blot analysis and immunofluorescence analysis. Conclusions Our study suggests that association of NEU2 with actin filaments and other protein(s) could be important for understanding the biological role of NEU2 in mammalian cells.

scholarly journals Large-scale identification of protein histidine methylation in human cells

2021 ◽  
Vol 3 (2) ◽  
Author(s):  
Sebastian Kapell ◽  
Magnus E Jakobsson
Keyword(s):  
Large Scale ◽  
Human Cells ◽  
System Level ◽  
Mass Spectrometry Analysis ◽  
Regulatory Function ◽  
Lysine Methylation ◽  
Spectrometry Analysis ◽  
Arginine Residues ◽  
Sequence Requirements ◽  
Level Analysis

Abstract Methylation can occur on histidine, lysine and arginine residues in proteins and often serves a regulatory function. Histidine methylation has recently attracted attention through the discovery of the human histidine methyltransferase enzymes SETD3 and METTL9. There are currently no methods to enrich histidine methylated peptides for mass spectrometry analysis and large-scale studies of the modification are hitherto absent. Here, we query ultra-comprehensive human proteome datasets to generate a resource of histidine methylation sites. In HeLa cells alone, we report 299 histidine methylation sites as well as 895 lysine methylation events. We use this resource to explore the frequency, localization, targeted domains, protein types and sequence requirements of histidine methylation and benchmark all analyses to methylation events on lysine and arginine. Our results demonstrate that histidine methylation is widespread in human cells and tissues and that the modification is over-represented in regions of mono-spaced histidine repeats. We also report colocalization of the modification with functionally important phosphorylation sites and disease associated mutations to identify regions of likely regulatory and functional importance. Taken together, we here report a system level analysis of human histidine methylation and our results represent a comprehensive resource enabling targeted studies of individual histidine methylation events.

Changes between longissimus dorsi muscle from donkey, cow, and goat assessed by transcriptomic and proteomic analyses

2020 ◽  
Author(s):  
Yan Sun ◽  
Jinpeng Wang ◽  
Yuhua Li ◽  
Chunhong Yang ◽  
Xiuge Wang ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fiber ◽  
Proteomic Analysis ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Expression Profiles ◽  
Differentially Expressed ◽  
Farm Animals ◽  
Meat Production ◽  
Altered Expression

Abstract Background: Domestic donkeys (Equus asinus) are farm animals; they are used mainly for carrying loads in the past centuries. Recently, interests on donkey milk and meat production have increased in many countries. Donkey meat is extremely popular in China due to its high nutritional value and unique flavor. Except the origin and domestication of donkeys, few genetic studies have been conducted. Moreover, data from transcriptional profiling and microRNA (miRNA) regulation of skeletal muscle tissues in donkey are scarce. Recent developments in high-throughput sequencing techniques can offer large-scale analysis of gene expression in different species. This study aimed to explore the differences of the molecular and regulation mechanisms among donkey meat, beef, and mutton using genome-wide transcriptomic analysis and proteomic methods to provide more effective genetic information.Methods: RNA sequencing and proteomic analysis (on the basis of isobaric tags for relative and absolute quantitation) on donkey, cow, and goat muscles, and miRNAs in donkey muscles were detected. We performed a comprehensive research on total RNA, including miRNAs from donkey muscles. The mRNA expression profiles were characterized and differences in single-copy homologous genes among species were analyzed.Results: Most differentially expressed genes were associated with pathways related to protein and fat synthesis and metabolism, muscle formation, and development. We identified single nucleotide polymorphisms (SNPs) in donkey muscle and alternative splicing (AS) events. A total of 57,201 putative SNPs were found, and the main SNP variations were located in known genes. Several AS events occurred in genes related to muscle fiber. Different AS events were also noted among species. Muscle proteomic data were obtained, including all expressed proteins and differentially expressed proteins. Combined transcriptomic and proteomic analysis effectively revealed pivotal mRNAs and proteins in muscles. We found five genes associated with thin and thick filaments, which indirectly explained the characteristics of donkey muscle fibers.Conclusion: RNA-seq and iTRAQ analysis revealed altered expression of genes and proteins in three kinds of muscle. In the highly expressed genes of donkey muscle, we discovered 31 expressed genes were involved in muscle contraction and skeletal muscle fiber development. Compared with mutton, five genes related to muscle fiber synthesis were found and showed differences both at transcriptional and proteome levels in donkey muscle. Meanwhile, genetic variation and regulatory factors can combine as a database to provide more valuable molecular information for further analysis.

scholarly journals Autoimmune gait disturbance accompanying adaptor protein-3B2-IgG

Neurology ◽  
2019 ◽  
Vol 93 (10) ◽  
pp. e954-e963 ◽  
Author(s):  
Josephe A. Honorat ◽  
A. Sebastian Lopez-Chiriboga ◽  
Thomas J. Kryzer ◽  
Lars Komorowski ◽  
Madeleine Scharf ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Western Blot ◽  
Adaptor Protein ◽  
Sensory Neuropathy ◽  
Clinical Information ◽  
Sympathetic Ganglia ◽  
Immunofluorescence Assay ◽  
Mass Spectrometry Analysis ◽  
Antigen Specificity ◽  
Spectrometry Analysis

ObjectiveTo describe phenotypes, treatment response, and outcomes of autoimmunity targeting a synaptic vesicle coat protein, the neuronal (B2) form of adaptor protein–3 (AP3).MethodsArchived serum and CSF specimens (from 616,025 screened) harboring unclassified synaptic antibodies mimicking amphiphysin–immunoglobulin G (IgG) on tissue-based indirect immunofluorescence assay (IFA) were re-evaluated for novel IgG staining patterns. Autoantigens were identified by western blot and mass spectrometry. Recombinant western blot and cell-binding assay (CBA) were used to confirm antigen specificity. Clinical data were obtained retrospectively.ResultsSerum (10) and CSF (6) specimens of 10 patients produced identical IFA staining patterns throughout mouse nervous system tissues, most prominently in cerebellum (Purkinje neuronal perikarya, granular layer synapses, and dentate regions), spinal cord gray matter, dorsal root ganglia, and sympathetic ganglia. The antigen revealed by mass spectrometry analysis and confirmed by recombinant assays (western blot and CBA) was AP3B2 in all. Of 10 seropositive patients, 6 were women; median symptom onset age was 42 years (range 24–58). Clinical information was available for 9 patients, all with subacute onset and rapidly progressive gait ataxia. Neurologic manifestations were myeloneuropathy (3), peripheral sensory neuropathy (2), cerebellar ataxia (2), and spinocerebellar ataxia (2). Five patients received immunotherapy; none improved, but they did not worsen over the follow-up period (median 36 months; range 3–94). Two patients (both with cancer) died. One of 50 control sera was positive by western blot only (but not by IFA or CBA).ConclusionAP3B2 (previously named β-neuronal adaptin-like protein) autoimmunity appears rare, is accompanied by ataxia (sensory or cerebellar), and is potentially treatable.

Application of IOT (Internet of Things) Technology in Chemical Testing with Large-Scale Analysis Instruments

2014 ◽  
Vol 701-702 ◽  
pp. 475-479
Author(s):  
Hong Bo Chen ◽  
Yi Zheng ◽  
Yan Gang Han
Keyword(s):  
Large Scale ◽  
Mass Spectrometry Analysis ◽  
Testing System ◽  
Spectrometry Analysis ◽  
Whole Process ◽  
Large Scale Analysis ◽  
Intelligent Methods ◽  
Internet Of Things Technology ◽  
Chemical Testing ◽  
Standard Development

In the field of chemical testing, it is of great importance to improve the accuracy and efficiency and reduce the risk caused by artificial factor with modern intelligent methods, which are critical to the standard development of testing labs. In this paper, intelligent control during the whole process of chromatography and mass spectrometry analysis with cloud computing technology was realized and discussed in detail. The intelligent testing system could be applied to the chemical analysis labs and spread to other fields.

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