30 research outputs found
Circulating Microvesicles from Pancreatic Cancer Accelerate the Migration and Proliferation of PANC‑1 Cells
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
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
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
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
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.
<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
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.
<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).
<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
Comparison of staining score of CD90 and CD24 expression between PanIN and normal pancreas.
<p>A: CD90 expression; B: CD24 expression. *means <i>P</i><0.05;**means <i>P</i><0.01.</p