Urinary Proteome Profiling Using 2D-DIGE and LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 299

are aligned on Typhoon scanner and photomultiplier tube
(PMT) voltage set to low (500 V) on each channel (Cy2,
Cy3 and Cy5).
(2) A preliminary low-resolution scan (1000 m) for each

channel is performed and grey scale pixel values generated.
Using ImageQuant software (GE Healthcare) for Typhoon
9400, maximum pixel values in various user-defined, spot-
rich regions of each image are obtained by adjusting PMT
voltages. Repeated scans may be required to ensure voltage
settings achieve maximum pixel values within 10% for each
of the three channels.
(3) High-resolution scans (100

μ

m) are then performed across
all gels at these optimal settings.

μ

(1) Scanned images are analysed using differential analysis soft-
ware (DeCyder version 5.1, later versions available). Spot
boundaries are defined using DeCyder differential in gel
analysis (DIA) module, allowing for intra-gel analysis, and
standardised abundance for each spot is obtained by com-
paring Cy3- and Cy5-labelled spots to the internal standard
(ratios - Cy3:Cy2 and Cy5:Cy2).
(2) Test spot volumes across all gels (inter-gel analysis) are
matched using the DeCyder biological variation analy-
sis (BVA) module. Lists of statistically significant deregu-
lated protein spots between disease and control samples
are generated using Student's t test or one- or two-way
ANOVA.
(3) BVA software is used to define the position of the refer-
ence markers and produce x

3.3.5. ImageAnalysis

y coordinates of each spot of
interest; these can be exported as a 'coordinate pick list.'

−

Post-staining of 2D gels using CCB is compatible with Cy dye
labelling and mass spectrometry and allows for improved spot
picking ( 18 ) . Figure 18.1 shows a post-stained CCB gel image
with differentially expressed and identified protein spots in a 2D-
DIGE comparison of pooled pancreatic ductal adenocarcinoma,
chronic pancreatitis and healthy urine specimens.

3.3.6.Post-staining
(andSpotPicking)

(1) After image capture gel plates are separated, individ-
ual gels bonded to low-fluorescent glass back plates are
washed, fixed and stained with GelCode Blue Safe Protein
Stain.
(2) GelCode Blue Safe Protein Stain (400 ml/gel) is added
to the washed gels in a new plastic tray and left shaking
for several hours to overnight at room temperature ( see
Note 14 ).

3.3.7.CCBPost-staining