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| Institute: |
Lab or Branch |
| NIDDK |
LBC |
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| Title: |
| Proteomics on secretory
proteins from adipose cells |
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| Authors: |
| X. Chen, S. W. Cushman,
S. Hess |
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| Abstract: |
| Recently, the critical
endocrine function of adipose cells to release signaling
molecules has been recognized. It is now known that adipose
cells play a central role in the pathogenesis of obesity
and its related diseases such as type II diabetes,
cardiovascular
disease, and certain cancers by secreting endocrine and
autocrine/paracrine factors. Dysregulation of adipose
cell secretion may be the initial defect responsible for
the development of disorders in other tissues/organs.
However, the signaling cascades are not well understood
and cannot be completely explained with the currently
known adipose-derived factors. Therefore, we have developed
a 2D-LC-MS/MS approach to cataloging secretory proteins
from adipose cells. This approach allowed us to catalog
a large number of proteins from different categories.
Adipose cells were isolated from rats and cultured in
a serum-free DMEM medium for 48 h. Culture medium was
concentrated and separated on a Zorbax C3 column using
a gradient from an aqueous solution of 0.1%TFA to acetonitrile.
Up to 60 fractions were collected and pooled to give 8
combined fractions. Each fraction was first reduced and
alkylated, then digested with Lys-C, followed by a tryptic
digest. The samples were separated on a C18 column. Peptides
were characterized by LC-MS/MS using a QTOF 2 mass
spectrometer.
Data were analyzed using a MASCOT search engine. From
comparison to adipose tissue stromal-vascular cells known
to contain populations of adipose cell precursors, we
could clearly show that mature adipose cells function
as secretory cells, and a large number of secreted proteins
was found. To those few proteins that are already known
to be secreted by adipose cells, we have compiled a group
of known secretory proteins previously not associated
with adipose cells and another group of unknown proteins.
According to their known functions, proteins were classified
into several categories including pro-inflammatory cytokines;
coagulation and complement factors; proteases/protease
inhibitors; proteins related to cardiovascular diseases,
reproduction, cell growth, and differentiation. Taken
together, we have been able to identify the secretory
proteome of adipose cells. This will allow us to develop
models of adipose cell signaling cascades, and lead to
an improved understanding of the biology of adipose cells
and the role of adipose cells in several pathophysiological
states. |
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