scholarly journals Comparative proteomic profiling of nuclear and cytosolic fractions from cell lines of different origin

2018 ◽  
Vol 64 (3) ◽  
pp. 233-240 ◽  
Author(s):  
I.V. Vakhrushev ◽  
S.E. Novikova ◽  
A.V. Tsvetkova ◽  
M.A. Pyatnitskiy ◽  
K.N. Yarygin
Keyword(s):  
Cell Lines ◽  
Proteomic Analysis ◽  
Biological Properties ◽  
Individual Characteristics ◽  
Mass Spectrometry Analysis ◽  
Nuclear Fraction ◽  
Proteomic Profiling ◽  
Spectrometry Analysis ◽  
Cellular Processes ◽  
Tissue Lysate

Proteomic analysis of the nuclear fraction is of great importance, since many cellular processes are initiated in the nucleus. Refinement and choice of experimental procedures for cell lysate fractionation and parameters for mass spectrometric detection and data processing continue to be of current interest. The mass spectrometry analysis presented here was tested on human cell lines derived from different tissues: HL-60 (peripheral blood); HepG2 (liver); EA.hy926 (vascular endothelium). High reproducibility of results and their consistency with biological properties of the objects under study were demonstrated. The use of cells of different types made it possible to reveal a set of 16 proteins whose LFQ-values allow for the discrimination between proteome fractions regardless of cell origin. Also, a set of 16 proteins is suggested which are associated with individual characteristics of cell lines regardless of cell fraction. These protein panels can serve as parameters to verify the proteomic analysis done was of sufficient quality, in particular as indicators of successful fractionation of cell or tissue lysate.

scholarly journals Direct-Tissue SELDI-TOF Mass Spectrometry Analysis: A New Application for Clinical Proteomics

2006 ◽  
Vol 52 (11) ◽  
pp. 2103-2106 ◽  
Author(s):  
Ali Bouamrani ◽  
Jessica Ternier ◽  
David Ratel ◽  
Alim-Louis Benabid ◽  
Jean-Paul Issartel ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Proteomic Analysis ◽  
Human Cancer ◽  
Clinical Proteomics ◽  
Mass Spectrometry Analysis ◽  
Mouse Tissue ◽  
Cancer Tissue ◽  
Proteomic Profiling ◽  
Spectrometry Analysis ◽  
Tof Ms

Abstract Background: New molecular profiling technologies can aid in analysis of small pathologic samples obtained by minimally invasive biopsy and may enable the discovery of key biomarkers synergistic with anatomopathologic analysis related to prognosis, therapeutic response, and innovative target validation. Thus proteomic analysis at the histologic level in healthy and pathologic settings is a major issue in the field of clinical proteomics. Methods: We used surface-enhanced laser desorption ionization-time-of-flight mass spectrometry (SELDI-TOF MS) technology with surface chromatographic subproteome enrichment and preservation of the spatial distribution of proteomic patterns to detect discrete modifications of protein expression. We performed in situ proteomic profiling of mouse tissue and samples of human cancer tissue, including brain and lung cancer. Results: This approach permitted the discrimination of glioblastomas from oligodendrogliomas and led to the identification of 3 potential markers. Conclusion: Direct tissue proteomic analysis is an original application of SELDI-TOF MS technology that can expand the use of clinical proteomics as a complement to the anatomopathological diagnosis.

scholarly journals CBMT-07. BIOMARKERS OF AGGRESSIVENESS IN IDH MUTANT GLIOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi34-vi34
Author(s):  
Tomohiro Yamasaki ◽  
Adrian Lita ◽  
Tyrone Dowdy ◽  
Mark Gilbert ◽  
Mioara Larion
Keyword(s):  
Cell Lines ◽  
Imaging Spectroscopy ◽  
Mutant Gene ◽  
Raman Imaging ◽  
Malignant Progression ◽  
Mass Spectrometry Analysis ◽  
Live Cells ◽  
Lower Grade ◽  
Spectrometry Analysis ◽  
Idh Mutations

Abstract BACKGROUND Gliomas with isocitrate dehydrogenase (IDH) mutations in adults evolve from lower-grade gliomas to secondary glioblastomas (GBM), a fatal disease with fast progression. IDH mutation occurs early in tumorigenesis, and persistently contribute to the reprograming of glucose, lipid and amino acid metabolism. This offer a plethora of potential biomarkers of progression. However, because it is extremely difficult to detect the distribution and transfer of metabolites changing in every moment in a single cell, the involvement of metabolites produced by mutant IDH in malignant progression remains understudied. MATERIALS AND METHODS Raman imaging spectroscopy, which can image chemical bonds and concentration of molecules at submicron spatial resolution, enables detection of spatiotemporal changes of metabolomes in live cells. We developed the software called Biomolecular Component Analysis (BCAbox) to deconvolute the recorded raw Raman spectra, leading to detection of unique spectral features of different classes of biomolecules. RESULTS AND CONCLUSIONS We applied Raman imaging spectroscopy to GBM cell lines that were transfected with IDH1 mutant gene. Our results indicated that lipid metabolism has a unique profile in IDH1 mutant gliomas. Subsequent mass spectrometry analysis of extracted organelle revealed the exact classes of lipids altered in the IDH mutant glioma and suggested biomarkers unique to IDH1 mutant. We will report our validation studies of the biomarkers in patient-derived IDH mutant glioma cell lines and patients derived-orthotopic xenograft mouse models with different degrees of aggressiveness and in matched primary versus recurrent gliomas. The results of the present study may provide novel insights into the discovery of metabolic biomarkers for the malignant progression in IDH mutant gliomas.

scholarly journals Thiol Peroxidase Is an Important Component of Streptococcus pneumoniae in Oxygenated Environments

2012 ◽  
Vol 80 (12) ◽  
pp. 4333-4343 ◽  
Author(s):  
Barak Hajaj ◽  
Hasan Yesilkaya ◽  
Rachel Benisty ◽  
Maayan David ◽  
Peter W. Andrew ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Mass Spectrometry Analysis ◽  
Activity Levels ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Cellular Processes ◽  
Thiol Peroxidase ◽  
Fine Tune

ABSTRACTStreptococcus pneumoniaeis an aerotolerant Gram-positive bacterium that causes an array of diseases, including pneumonia, otitis media, and meningitis. During aerobic growth,S. pneumoniaeproduces high levels of H2O2. SinceS. pneumoniaelacks catalase, the question of how it controls H2O2levels is of critical importance. Thepsalocus encodes an ABC Mn2+-permease complex (psaBCA) and a putative thiol peroxidase,tpxD. This study shows thattpxDencodes a functional thiol peroxidase involved in the adjustment of H2O2homeostasis in the cell. Kinetic experiments showed that recombinant TpxD removed H2O2efficiently. However,in vivoexperiments revealed that TpxD detoxifies only a fraction of the H2O2generated by the pneumococcus. Mass spectrometry analysis demonstrated that TpxD Cys58undergoes selective oxidationin vivo, under conditions where H2O2is formed, confirming the thiol peroxidase activity. Levels of TpxD expression and synthesisin vitrowere significantly increased in cells grown under aerobic versus anaerobic conditions. The challenge with D39 and TIGR4 with H2O2resulted intpxDupregulation, whilepsaBCAexpression was oppositely affected. However, the challenge of ΔtpxDmutants with H2O2did not affectpsaBCA, implying that TpxD is involved in the regulation of thepsaoperon, in addition to its scavenging activity. Virulence studies demonstrated a notable difference in the survival time of mice infected intranasally with D39 compared to that of mice infected intranasally with D39ΔtpxD. However, when bacteria were administered directly into the blood, this difference disappeared. The findings of this study suggest that TpxD constitutes a component of the organism's fundamental strategy to fine-tune cellular processes in response to H2O2.

scholarly journals Kalirin Interacts with TRAPP and Regulates Rab11 and Endosomal Recycling

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1132
Author(s):  
Xiaolong Wang ◽  
Meiqian Weng ◽  
Yuting Ke ◽  
Ellen Sapp ◽  
Marian DiFiglia ◽  
...  
Keyword(s):  
Low Frequency ◽  
Cell Movement ◽  
Mass Spectrometry Analysis ◽  
Nucleotide Exchange ◽  
Vesicle Budding ◽  
Recycling Endosomes ◽  
Spectrometry Analysis ◽  
Cellular Processes ◽  
Nucleotide Exchange Factor ◽  
Elevated Expression

Coordinated actions of Rab and Rho are necessary for numerous essential cellular processes ranging from vesicle budding to whole cell movement. How Rab and Rho are choreographed is poorly understood. Here, we report a protein complex comprised of kalirin, a Rho guanine nucleotide exchange factor (GEF) activating Rac1, and RabGEF transport protein particle (TRAPP). Kalirin was identified in a mass spectrometry analysis of proteins precipitated by trappc4 and detected on membranous organelles containing trappc4. Acute knockdown of kalirin did not affect trappc4, but significantly reduced overall and membrane-bound levels of trappc9, which specifies TRAPP toward activating Rab11. Trappc9 deficiency led to elevated expression of kalirin in neurons. Co-localization of kalirin and Rab11 occurred at a low frequency in NRK cells under steady state and was enhanced upon expressing an inactive Rab11 mutant to prohibit the dissociation of Rab11 from the kalirin-TRAPP complex. The small RNA-mediated depletion of kalirin diminished activities in cellular membranes for activating Rab11 and resulted in a shift in size of Rab11 positive structures from small to larger ones and tubulation of recycling endosomes. Our study suggests that kalirin and TRAPP form a dual GEF complex to choreograph actions of Rab11 and Rac1 at recycling endosomes.

scholarly journals Jacobsen Catalyst as a Cytochrome P450 Biomimetic Model for the Metabolism of Monensin A

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Bruno Alves Rocha ◽  
Anderson Rodrigo Moraes de Oliveira ◽  
Murilo Pazin ◽  
Daniel Junqueira Dorta ◽  
Andresa Piacezzi Nascimento Rodrigues ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Liver Mitochondria ◽  
Biological Properties ◽  
Model Systems ◽  
Mass Spectrometry Analysis ◽  
Rat Liver Mitochondria ◽  
Model Assessment ◽  
Spectrometry Analysis ◽  
Monensin A

Monensin A is a commercially important natural product isolated fromStreptomyces cinnamonensinsthat is primarily employed to treat coccidiosis. Monensin A selectively complexes and transports sodium cations across lipid membranes and displays a variety of biological properties. In this study, we evaluated the Jacobsen catalyst as a cytochrome P450 biomimetic model to investigate the oxidation of monensin A. Mass spectrometry analysis of the products from these model systems revealed the formation of two products: 3-O-demethyl monensin A and 12-hydroxy monensin A, which are the same ones found inin vivomodels. Monensin A and products obtained in biomimetic model were tested in a mitochondrial toxicity model assessment and an antimicrobial bioassay againstStaphylococcus aureus, S. aureusmethicillin-resistant,Staphylococcus epidermidis, Pseudomonas aeruginosa,andEscherichia coli.Our results demonstrated the toxicological effects of monensin A in isolated rat liver mitochondria but not its products, showing that the metabolism of monensin A is a detoxification metabolism. In addition, the antimicrobial bioassay showed that monensin A and its products possessed activity against Gram-positive microorganisms but not for Gram-negative microorganisms. The results revealed the potential of application of this biomimetic chemical model in the synthesis of drug metabolites, providing metabolites for biological tests and other purposes.

scholarly journals Functional Characterization of Sesquiterpene Synthase fromPolygonum minus

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Su-Fang Ee ◽  
Zeti-Azura Mohamed-Hussein ◽  
Roohaida Othman ◽  
Noor Azmi Shaharuddin ◽  
Ismanizan Ismail ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Metal Binding ◽  
Functional Characterization ◽  
Biological Properties ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Sesquiterpene Synthase ◽  
Conserved Motifs ◽  
Spectrometry Analysis ◽  
Metal Binding Domain

Polygonum minusis an aromatic plant, which contains high abundance of terpenoids, especially the sesquiterpenes C15H24. Sesquiterpenes were believed to contribute to the many useful biological properties in plants. This study aimed to functionally characterize a full length sesquiterpene synthase gene fromP. minus.P. minussesquiterpene synthase (PmSTS) has a complete open reading frame (ORF) of 1689 base pairs encoding a 562 amino acid protein. Similar to other sesquiterpene synthases, PmSTS has two large domains: the N-terminal domain and the C-terminal metal-binding domain. It also consists of three conserved motifs: the DDXXD, NSE/DTE, and RXR. A three-dimensional protein model for PmSTS built clearly distinguished the two main domains, where conserved motifs were highlighted. We also constructed a phylogenetic tree, which showed that PmSTS belongs to the angiosperm sesquiterpene synthase subfamily Tps-a. To examine the function ofPmSTS, we expressed this gene inArabidopsis thaliana. Two transgenic lines, designated asOE3andOE7, were further characterized, both molecularly and functionally. The transgenic plants demonstrated smaller basal rosette leaves, shorter and fewer flowering stems, and fewer seeds compared to wild type plants. Gas chromatography-mass spectrometry analysis of the transgenic plants showed that PmSTS was responsible for the production ofβ-sesquiphellandrene.

scholarly journals Temporal Quantitative Phosphoproteomics Profiling of Interleukin-33 Signaling Network Reveals Unique Modulators of Monocyte Activation

Cells ◽  
2022 ◽  
Vol 11 (1) ◽  
pp. 138
Author(s):  
Devasahayam Arokia Balaya Rex ◽  
Yashwanth Subbannayya ◽  
Prashant Kumar Modi ◽  
Akhina Palollathil ◽  
Lathika Gopalakrishnan ◽  
...  
Keyword(s):  
Inflammatory Diseases ◽  
Leukocyte Adhesion ◽  
Cell Cycle Checkpoints ◽  
Mass Spectrometry Analysis ◽  
Dna Damage And Repair ◽  
Spectrometry Analysis ◽  
Interleukin 33 ◽  
Cellular Processes ◽  
Defensive Role ◽  
Signaling Modules

Interleukin-33 (IL-33), a member of the IL-1 superfamily cytokines, is an endogenous danger signal and a nuclear-associated cytokine. It is one of the essential mediators of both innate and adaptive immune responses. Aberrant IL-33 signaling has been demonstrated to play a defensive role against various infectious and inflammatory diseases. Although the signaling responses mediated by IL-33 have been previously reported, the temporal signaling dynamics are yet to be explored. To this end, we applied quantitative temporal phosphoproteomics analysis to elucidate pathways and proteins induced by IL-33 in THP-1 monocytes. Employing a TMT labeling-based quantitation and titanium dioxide (TiO2)-based phosphopeptide enrichment strategy followed by mass spectrometry analysis, we identified and quantified 9448 unique phosphopeptides corresponding to 3392 proteins that showed differential regulation. Of these, 171 protein kinases, 60 phosphatases and 178 transcription factors were regulated at different phases of IL-33 signaling. In addition to the confirmed activation of canonical signaling modules including MAPK, NFκB, PI3K/AKT modules, pathway analysis of the time-dependent phosphorylation dynamics revealed enrichment of several cellular processes, including leukocyte adhesion, response to reactive oxygen species, cell cycle checkpoints, DNA damage and repair pathways. The detailed quantitative phosphoproteomic map of IL-33 signaling will serve as a potentially useful resource to study its function in the context of inflammatory and pathological conditions.

scholarly journals Rules for PP2A-controlled phosphosignalling and drug responses

2018 ◽  
Author(s):  
Otto Kauko ◽  
Susumu Y. Imanishi ◽  
Evgeny Kulesskiy ◽  
Teemu Daniel Laajala ◽  
Laxman Yetukuri ◽  
...  
Keyword(s):  
Cancer Cell ◽  
Mass Spectrometry Analysis ◽  
Drug Responses ◽  
Spectrometry Analysis ◽  
Cellular Processes ◽  
Cell Responses ◽  
Systemic Analysis ◽  
Phosphatase 2A ◽  
Cancer Drivers ◽  
Systemic Understanding

AbstractSystemic understanding of protein phosphatase 2A (PP2A)-regulated cellular processes is still at infancy. Here, we present mass-spectrometry analysis of phospho-targets (dephosphorylome) regulated by PP2A modulation. In addition to PP2A-regulated processes and targets, the data reveal important general concepts and rules related to PP2A-mediated phosphoregulation. These include the unidirectionality paradigm of regulation of phosphorylation, and differential spatial distribution of kinase-and phosphatase-dominated phosphotargets. Data also present first systemic analysis of targets of PP2A-modulating oncoproteins, CIP2A, PME-1, and SET; including targets via which PP2A may coordinately regulate activities of cancer drivers and tumor suppressors such as MYC or TP53. To validate functional utility of this dataset, PP2A dephosphorylome activity was correlated with cancer cell responses to over 300 drugs. Notably, we find that cancer therapy responses can be broadly classified based on PP2A dephosphorylome activity, both in quantitative and qualitative manner. In summary, our data characterize rules by which PP2A coordinate cancer cell phosphosignaling and drug responses. The results also may also direct the use of emerging pharmacological approaches for PP2A activity modulation in human diseases.

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