Biology Reference
In-Depth Information
microglia to the inflammatory responses was assessed by measuring nitric oxide
(NO) and tumor necrosis factor (TNF)- production following 72 hours of cell
exposure to 10 g/ml lipopolysaccharide (LPS)
[45]
. Only LPS-unresponsive astro-
cyte cultures were used in further experiments. Twenty-four hours before treat-
ments, cells were placed in fresh medium containing 2% fetal bovine serum (FBS).
Astrocytes were stimulated with 40 nM purified recombinant HMGB1 for 8 hours or
left untreated and lysed (as specified in
[46]
); 0.7 mg of each sample was submitted
to bidimensional (2D) liquid chromatography. Proteomic analysis was carried out by
using the ProteomeLab PF2D system (Beckman Coulter, Inc.), which separates pro-
teins according to isoelectric point by means of a chromatofocusing column in the
first dimension, and by means of nonporous reversed-phase (RP) high performance
liquid chromatography (HPLC) in the second dimension. The first-dimension method
was started at a flow rate of 0.2 ml/min by washing the column with 100% start buffer
for 45 minutes; 1 ml fractions were collected at 5-minute intervals. The linear pH gra-
dient was generated by running 100% eluent buffer (pH 4.5 0.1; Beckman Coulter,
Inc.) for 1 hour; fractions were collected every 0.3 pH units. When the eluent reached
pH 4.5, the column was washed with 10 volumes of 1 M NaCl to remove residual
proteins. The fractions generated in the first dimension were sequentially injected
(250 l) onto the second-dimension RP HPLC column. The second-dimension method
consisted of a 30-minute linear gradient of 5-100% B/A at 0.75 ml/min, where A is
0.10% trifluoroacetic acid (TFA) in water and B is 0.08% TFA in acetonitrile. Data
were collected and analyzed by means of ProteoVue/DeltaVue software (Beckman
Coulter, Inc.). ProteoVue allows comparison of all second-dimension runs for each
sample in a banded map display that represents the UV peak intensity, where each
band is a pH fraction. DeltaVue allows side-by-side viewing of the second-dimension
runs for two samples. DeltaVue also shows the difference map between two samples
from their ProteoVue maps.
Figure 3.1
shows the map of HMGB1 up-regulated
proteins obtained by comparing the proteomic profile of untreated and of HMGB1-
stimulated astrocytes. The map reports the pH gradient versus RP retention time. The
band intensity is proportional to the UV absorbance of each protein peak.
At first, we started to analyze protein peaks up-regulated in HMGB1-stimulated
astrocytes. Proteins that increased more than twofold were selected for further mass
spectrometry analysis and peptide mass fingerprint procedure
[46]
. Nine of the up-
regulated proteins, indicated as numbered spots in
Figure 3.1
, were identified using
a MASCOT search engine (
http://
www.matrixscience.com
)
. One missed cleavage
per peptide was allowed, and an initial mass tolerance of 50 ppm was adopted. If
the proteins were not significantly identified, the mass tolerance was extended to
100 ppm. As shown in
Table 3.1
, HMGB1 induced a threefold increase in the levels
of phosphorylated glial fibrillary acidic protein (GFAP) and vimentin, two glial inter-
mediate filament proteins commonly accepted as astrogliosis parameters
[47,48]
.
HMGB1 also promoted a 2.5-fold up-regulation of alpha enolase, one of the major
proteins previously found to be overexpressed in astrocytes stimulated with a com-
plete cytokine mixture (CCM)
[43]
. Among the other major proteins up-regulated by
HMGB1, only heat shock protein (HSP)10 has previously been related to inflamma-
tory processes
[49]
.
