Biology Reference
In-Depth Information
5. Calibration is performed manually for the less than 1% samples for which
automatic calibration fails. After calibration, the peak lists are created using the
“peak to mascot” script of the 4700 Explorer™ software with the following
settings: mass range from 900 to 3700 Da, peak density of 50 peaked per range
of 200 Da, minimal area of 100 and maximal 200 peaks per protein spot, and
minimal S/N ratio of 6. The resulting peak lists are compared with organism-
specific sequence databases by using the mascot search engine (Matrix Science,
London, UK). Peptide mixtures that yield at least twice a Mowse score of at least
49 (depending on the size and quality of the database) and sequence coverage of
at least 30% are regarded as positive identifications.
6. Proteins that fail to exceed the 30% sequence coverage cut-off are subjected
to MALDI-MS/MS. MALDI-TOF-TOF analysis is performed for the three
strongest peaks of the TOF spectrum. For one main spectrum, 20 subspectra
with 125 shots per subspectrum are accumulated using a random search pattern.
The internal calibration is automatically performed as one-point calibration if the
monoisotopic arginine (M+H) + m/z at 175.119 or lysine (M+H) + m/z at 147.107
reaches an S/N of at least 5. The peak lists are created using the “peak to mascot”
script of the 4700 Explorer™ software with the following settings: mass range
from 60 Da to a mass that was 20 Da lower than the precursor mass, peak
density of five peaks per 200 Da, minimal area of 100 and maximal 20 peaks per
precursor, and a minimal S/N ratio of 5. Database searches are performed using
the GPS explorer software with the organism-specific databases. Proteins with a
Mowse score of at least 49 in the reflector mode that is confirmed by subsequent
peptide/fragment identifications of the strongest peaks (MS/MS) are regarded as
identified. MS/MS analysis is particularly useful for the identification of spots
containing more than one protein.
3.7. Quantitation and Bioinformatic Approaches
The 2D gel image analysis is performed with the Delta2D Software (Decodon
GmbH, Greifswald, Germany). Three different data sets of each experiment
have to be analyzed in order to screen for differences in the amount or synthesis
of the proteins identified on 2D gels.
The dual-channel imaging technique is an excellent tool for identifying
all proteins induced or repressed by growth-restricting stimuli (15) . In this
technique, two digitized images of 2D gels are generated and combined in
alternate additive dual-color channels ( see Fig. 5 and Color Plate 3, following
p. 46). The first image (densitogram), which shows protein levels visualized
by various staining techniques, is false-colored green. The second image
(autoradiograph), representing proteins synthesized and radioactively labeled
during a 5-min pulse labeling with [ 35 S]-l-methionine, is false-colored red.
When the two images are combined, proteins accumulated and synthesized in
growing cells are colored yellow. However, proteins not previously accumu-
lated in the cell but newly synthesized after the imposition of a stress or
 
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