Search results for "PROTEOMICS"
showing 10 items of 534 documents
Ultrahigh-Throughput Proteomics Using Fast RPLC Separations with ESI-MS/MS
2005
We describe approaches for proteomics analysis using electrospray ionization-tandem mass spectrometry coupled with fast reversed-phase liquid chromatography (RPLC) separations. The RPLC separations used 50-microm-i.d. fused-silica capillaries packed with submicrometer-sized C18-bonded porous silica particles and achieved peak capacities of 130-420 for analytes from proteome tryptic digests. When these separations were combined with linear ion trap tandem mass spectrometry measurements, approximately 1000 proteins could be identified in 50 min from approximately 4000 identified tryptic peptides; approximately 550 proteins in 20 min from approximately 1800 peptides; and approximately 250 prot…
Proteomic Profiling of Secreted Proteins for the Hematopoietic Support of Interleukin-Stimulated Human Umbilical Vein Endothelial Cells
2013
Human umbilical cord vein endothelial cells (HUVECs) secrete a number of factors that greatly impact the proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). These factors remain largely unknown. Here, we report on the most comprehensive proteomic profiling of the HUVEC secretome and identified 827 different secreted proteins. Two hundred and thirty-one proteins were found in all conditions, whereas 369 proteins were identified only under proinflammatory conditions following IL-1β, IL-3, and IL-6 stimulation. Thirteen proteins including complement factor b (CFb) were identified only under IL-1β and IL-3 conditions and may potentially represent HSPC prolifer…
Multiple changes induced by fibroblasts on breast cancer cells.
2010
It is now widely recognised that the cross-talk between cancer and stromal cells may play a crucial role in cancer progression. However little is known about the complex underlying molecular mechanisms that occur within the tumor microenvironment. Fibroblasts are the major stromal cells with multiple roles, especially towards both the extracellular matrix and the neighbouring cell population, including neoplastic cells. Consequently, proteomic analyses would provide a wider resource for a better understanding of the potential modulating effects exerted by fibroblasts on cancer cells. In this report we describe the effects of fibroblast stimulation on the breast cancer cell line (8701-BC) pr…
Surface proteomic analysis of differentiated versus stem-like osteosarcoma human cells.
2013
Cancer stem cell characterization represents a breakthrough in cancer research. Despite evidence showing the existence and the role of cancer stem cells in osteosarcoma (OS) onset and progression, little is known about their specific surface phenotype. To address this issue, we carried out a cytometric analysis with an antibody-array comprising 245 membrane proteins comparing the stem and differentiated OS cells. As experimental model, we chose the stem-like cell line 3aminobenzamide-OS and its parental, differentiated, cell line MG63. We identified 50 differentially expressed, 23 homogeneously expressed, and 172 not expressed proteins in the two cell line models, thus defining a surface pr…
Building high-quality assay libraries for targeted analysis of SWATH MS data
2015
Targeted proteomics by selected/multiple reaction monitoring (S/MRM) or, on a larger scale, by SWATH (sequential window acquisition of all theoretical spectra) MS (mass spectrometry) typically relies on spectral reference libraries for peptide identification. Quality and coverage of these libraries are therefore of crucial importance for the performance of the methods. Here we present a detailed protocol that has been successfully used to build high-quality, extensive reference libraries supporting targeted proteomics by SWATH MS. We describe each step of the process, including data acquisition by discovery proteomics, assertion of peptide-spectrum matches (PSMs), generation of consensus sp…
Membrane vesicles containing matrix metalloproteinase-9 and fibroblast growth factor-2 are released into the extracellular space from mouse mesoangio…
2010
Certain proteins, including fibroblast growth factor-2 (FGF-2) and matrix metalloproteinase-9 (MMP-9), have proved very effective in increasing the efficacy of mesoangioblast stem cell therapy in repairing damaged tissue. We provide the first evidence that mouse mesoangioblast stem cells release FGF-2 and MMP-9 in their active form through the production of membrane vesicles. These vesicles are produced and turned over continuously, but are stable for some time in the extracellular milieu. Mesoangioblasts shed membrane vesicles even under oxygen tensions that are lower than those typically used for cell culture and more like those of mouse tissues. These findings suggest that mesoangioblast…
Mutations in DMI3 and SUNN modify the appressorium-responsive root proteome in arbuscular mycorrhiza.
2006
Modification of the Medicago truncatula root proteome during the early stage of arbuscular mycorrhizal symbiosis was investigated by comparing, using two-dimensional electrophoresis, the protein patterns obtained from non-inoculated roots and roots synchronized for Glomus intraradices appressorium formation. This approach was conducted in wild-type (J5), mycorrhiza-defective (TRV25, dmi3), and autoregulation-defective (TR122, sunn) M. truncatula genotypes. The groups of proteins that responded to appressorium formation were further compared between wild-type and mutant genotypes; few overlaps and major differences were recorded, demonstrating that mutations in DMI3 and SUNN modified the ap…
Mitochondrial proteomics profile points oxidative phosphorylation as main target for beauvericin and enniatin B mixture
2020
Abstract Beauvericin (BEA) and enniatin B (EN B) are non-legislated Fusarium mycotoxins usually found in cereal and cereal-based products all around the world. By the proteomic analysis of mitochondria enriched extracts from Jurkat cells exposed for 24 h to three concentrations of BEA:EN B (0.01–0.1–0.5 μM), a number of 1821 proteins (202 mitochondrial) were identified and relatively quantified. 340 proteins (59 mitochondrial) were statistically significant altered in our samples (Anova p-value ≤ 0.05 and fold change (FC) ≥1.5). The protein mitochondrial translational release factor 1 like (MTRF1L) was the most abundant protein in the three mycotoxin exposures studied. The mycotoxins mixtur…
C7 is expressed on endothelial cells as a trap for the assembling terminal complement complex and may exert anti-inflammatory function.
2009
AbstractWe describe a novel localization of C7 as a membrane-bound molecule on endothelial cells (ECs). Data obtained by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis, Northern blot analysis, and mass spectrometry revealed that membrane-associated C7 (mC7) was indistinguishable from soluble C7 and was associated with vimentin on the cell surface. mC7 interacted with the other late complement components to form membrane-bound TCC (mTCC). Unlike the soluble SC5b-9, mTCC failed to stimulate ECs to express adhesion molecules, to secrete IL-8, and to induce albumin leakage through a monolayer of ECs, and more importantly protected ECs from the proinf…
The substrate degradome of meprin metalloproteases reveals an unexpected proteolytic link between meprin β and ADAM10
2012
The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and β we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)—the constitutive α-secretase—is activated by meprin β through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin…