30 research outputs found

    Circulating Microvesicles from Pancreatic Cancer Accelerate the Migration and Proliferation of PANC‑1 Cells

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    Circulating microvesicles are able to mediate long-distance cell–cell communications. It is essential to understand how microvesicles from pancreatic cancer act on other cells in the body. In this work, serum-derived microvesicles were isolated from 10 patients with locally advanced pancreatic cancer and healthy controls. Using Cell Transwell and WST-1 reagents, we found that microvesicles from pancreatic cancer accelerated migration and proliferation of PANC-1 cells. Meanwhile, the proliferation of these cancer-microvesicle-treated cells (CMTCs) was affected less by 10 μM of gemcitabine relative to healthy microvesicle-treated cells (HMTCs). Next, we optimized the filter-aided sample preparation method to increase the recovery of protein samples and then applied it to the quantification of the proteome of CMTCs and HMTCs. The peptides were labeled and analyzed by liquid chromatography–tandem mass spectrometry. In total, 4102 proteins were identified, where 35 proteins were up-regulated with 27 down-regulated in CMTCs. We verified the quantitative results of three key proteins CD44, PPP2R1A, and TP53 by Western blot. The Ingenuity Pathway Analysis revealed pathways that cancer microvesicles might participate in to promote cell migration and proliferation. These findings may provide novel clues of treatment for tumorigenesis and metastasis

    Circulating Microvesicles from Pancreatic Cancer Accelerate the Migration and Proliferation of PANC‑1 Cells

    No full text
    Circulating microvesicles are able to mediate long-distance cell–cell communications. It is essential to understand how microvesicles from pancreatic cancer act on other cells in the body. In this work, serum-derived microvesicles were isolated from 10 patients with locally advanced pancreatic cancer and healthy controls. Using Cell Transwell and WST-1 reagents, we found that microvesicles from pancreatic cancer accelerated migration and proliferation of PANC-1 cells. Meanwhile, the proliferation of these cancer-microvesicle-treated cells (CMTCs) was affected less by 10 μM of gemcitabine relative to healthy microvesicle-treated cells (HMTCs). Next, we optimized the filter-aided sample preparation method to increase the recovery of protein samples and then applied it to the quantification of the proteome of CMTCs and HMTCs. The peptides were labeled and analyzed by liquid chromatography–tandem mass spectrometry. In total, 4102 proteins were identified, where 35 proteins were up-regulated with 27 down-regulated in CMTCs. We verified the quantitative results of three key proteins CD44, PPP2R1A, and TP53 by Western blot. The Ingenuity Pathway Analysis revealed pathways that cancer microvesicles might participate in to promote cell migration and proliferation. These findings may provide novel clues of treatment for tumorigenesis and metastasis

    High-Performance Chemical Isotope Labeling Liquid Chromatography Mass Spectrometry for Exosome Metabolomics

    No full text
    Circulating exosomes in bodily fluids such as blood are being actively studied as a rich source of chemical biomarkers for cancer diagnosis and monitoring. Although nucleic acid analysis is a primary tool for the discovery of circulating biomarkers in exosomes, metabolomics holds the potential of expanding the chemical diversity of biomarkers that may be easy and rapid to detect. However, only trace amounts of exosomes can be isolated from a small volume of patient blood, and thus a very sensitive technique is required to analyze the metabolome of exosomes. In this report, we present a workflow that involves multiple cycles of ultracentrifugation for exosome isolation using a starting material of 2 mL of human serum, freeze–thaw-cycles in 50% methanol/water for exosome lysis and metabolite extraction, differential chemical isotope labeling (CIL) of metabolites for enhancing liquid chromatography (LC) separation and improving mass spectrometry (MS) detection, and nanoflow LC-MS (nLC-MS) with captivespray for analysis. As a proof-of-principle, we used dansylation labeling to analyze the amine- and phenol-submetabolomes in two sets of exosome samples isolated from the blood samples of five pancreatic cancer patients before and after chemotherapy treatment. The average number of peak pairs or metabolites detected was 1964 ± 60 per sample for a total of 2446 peak pairs (<i>n</i> = 10) in the first set and 1948 ± 117 per sample for a total of 2511 peak pairs (<i>n</i> = 10) in the second set. There were 101 and 94 metabolites positively identified in the first and second set, respectively, and 1580 and 1590 peak pairs with accurate masses matching those of metabolites in the MyCompoundID metabolome database. Analyzing the mixtures of <sup>12</sup>C-labeled individual exosome samples spiked with a <sup>13</sup>C-labeled pooled sample which served as an internal standard allowed relative quantification of metabolomic changes of exosomes of blood samples collected before and after treatment

    Citric Acid-Assisted Two-Step Enrichment with TiO<sub>2</sub> Enhances the Separation of Multi- and Monophosphorylated Peptides and Increases Phosphoprotein Profiling

    No full text
    Phosphopeptide enrichment is essential for large-scale phosphoprotein profiling. Titanium dioxide (TiO<sub>2</sub>) is widely used in phosphopeptide enrichment, but it is limited in the isolation of multiphosphorylated peptides due to their strong binding. In this study, we found that citric acid greatly affects the binding of mono- and multiphosphopeptides with TiO<sub>2</sub>, which can be used for stepwise phosphopeptide separation coupled with mass spectrum (MS) identification. We first loaded approximately 1 mg of peptide mixture of HeLa cell digests onto TiO<sub>2</sub> beads in highly concentrated citric acid (1 M). Then the flow-through fraction was diluted to ensure low concentration of citric acid (50 mM) and followed by loading onto another aliquot of TiO<sub>2</sub> beads. The two eluted fractions were subjected to nanoLC–MS/MS analysis. We identified 1,500 phosphorylated peptides, of which 69% were multiphosphorylated after the first enrichment. After the second enrichment, 2,167 phosphopeptides, of which 92% were monophosphorylated, were identified. In total, we successfully identified 3,136 unique phosphopeptides containing 3,973 phosphosites utilizing this strategy. Finally, more than 37% of the total phosphopeptides and 2.6-fold more of the multiphosphorylated peptides were identified as compared to the frequently used DHB/TiO<sub>2</sub> enrichment strategy. Combining SCX with CATSET, we identified 14,783 phosphopeptides and 15,713 phosphosites, of which 3,678 were unrecorded in PhosphoSitePlus database. This two-step separation procedure for sequentially enriching multi- and monophosphorylated peptides by using citric acid is advantageous in multiphosphorylated peptide separation, as well as for more comprehensive phosphoprotein profiling

    Citric Acid-Assisted Two-Step Enrichment with TiO<sub>2</sub> Enhances the Separation of Multi- and Monophosphorylated Peptides and Increases Phosphoprotein Profiling

    No full text
    Phosphopeptide enrichment is essential for large-scale phosphoprotein profiling. Titanium dioxide (TiO<sub>2</sub>) is widely used in phosphopeptide enrichment, but it is limited in the isolation of multiphosphorylated peptides due to their strong binding. In this study, we found that citric acid greatly affects the binding of mono- and multiphosphopeptides with TiO<sub>2</sub>, which can be used for stepwise phosphopeptide separation coupled with mass spectrum (MS) identification. We first loaded approximately 1 mg of peptide mixture of HeLa cell digests onto TiO<sub>2</sub> beads in highly concentrated citric acid (1 M). Then the flow-through fraction was diluted to ensure low concentration of citric acid (50 mM) and followed by loading onto another aliquot of TiO<sub>2</sub> beads. The two eluted fractions were subjected to nanoLC–MS/MS analysis. We identified 1,500 phosphorylated peptides, of which 69% were multiphosphorylated after the first enrichment. After the second enrichment, 2,167 phosphopeptides, of which 92% were monophosphorylated, were identified. In total, we successfully identified 3,136 unique phosphopeptides containing 3,973 phosphosites utilizing this strategy. Finally, more than 37% of the total phosphopeptides and 2.6-fold more of the multiphosphorylated peptides were identified as compared to the frequently used DHB/TiO<sub>2</sub> enrichment strategy. Combining SCX with CATSET, we identified 14,783 phosphopeptides and 15,713 phosphosites, of which 3,678 were unrecorded in PhosphoSitePlus database. This two-step separation procedure for sequentially enriching multi- and monophosphorylated peptides by using citric acid is advantageous in multiphosphorylated peptide separation, as well as for more comprehensive phosphoprotein profiling

    Representative images of CD90 (green) with CD45(red) both in PanIN III and PanIN III.

    No full text
    <p>Nuclei visualization was shown by staining DAPI (blue). CD45 was negative in PanINs. Scale bars = 100 μm.</p

    Citric Acid-Assisted Two-Step Enrichment with TiO<sub>2</sub> Enhances the Separation of Multi- and Monophosphorylated Peptides and Increases Phosphoprotein Profiling

    No full text
    Phosphopeptide enrichment is essential for large-scale phosphoprotein profiling. Titanium dioxide (TiO<sub>2</sub>) is widely used in phosphopeptide enrichment, but it is limited in the isolation of multiphosphorylated peptides due to their strong binding. In this study, we found that citric acid greatly affects the binding of mono- and multiphosphopeptides with TiO<sub>2</sub>, which can be used for stepwise phosphopeptide separation coupled with mass spectrum (MS) identification. We first loaded approximately 1 mg of peptide mixture of HeLa cell digests onto TiO<sub>2</sub> beads in highly concentrated citric acid (1 M). Then the flow-through fraction was diluted to ensure low concentration of citric acid (50 mM) and followed by loading onto another aliquot of TiO<sub>2</sub> beads. The two eluted fractions were subjected to nanoLC–MS/MS analysis. We identified 1,500 phosphorylated peptides, of which 69% were multiphosphorylated after the first enrichment. After the second enrichment, 2,167 phosphopeptides, of which 92% were monophosphorylated, were identified. In total, we successfully identified 3,136 unique phosphopeptides containing 3,973 phosphosites utilizing this strategy. Finally, more than 37% of the total phosphopeptides and 2.6-fold more of the multiphosphorylated peptides were identified as compared to the frequently used DHB/TiO<sub>2</sub> enrichment strategy. Combining SCX with CATSET, we identified 14,783 phosphopeptides and 15,713 phosphosites, of which 3,678 were unrecorded in PhosphoSitePlus database. This two-step separation procedure for sequentially enriching multi- and monophosphorylated peptides by using citric acid is advantageous in multiphosphorylated peptide separation, as well as for more comprehensive phosphoprotein profiling

    Expression of CD24 (red) in normal and various PanIN tissues.

    No full text
    <p>Nuclei visualization was shown by staining DAPI (blue). CD24 presented weak or negative expression in acinar cells, and no staining was observed in pancreatic ductal epithelium in the normal goup. CD24 was mainly present in the cytoplasm and membrane of the pancreatic ductal epithelium, especially in the apical epithelium of the duct (arrow). Scale bars = 100 μm.</p

    Three PanIN hispathological grades (HE).

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    <p>A:normal pancreas; B:PanIN III, minimal cytological atypia (arrow); C:PanIN III, nuclear polarity disappears, moderate cytological atypia (arrow); D: PanIN III, nuclear atypia hyperplasia, papillary or micro papillary morphology (arrow). Scale bars = 100 μm.</p
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