Proteomic Analysis to Investigate Regulatory Networks in Staphylococcus aureus - Bacterial Pathogenesis: Methods and Protocols - page 37

1. Place gels directly on the wet surface of the Typhoon.
2. For scanning, use the BLUE2-Laser (488 nm) for Cy2-labeled proteins, the green
laser (532 nm) for Cy3-labeled proteins, and the red laser (633 nm) for Cy5-labeled
proteins ( see Note 10 ).
3. For quantitation, scan the gels at 100 dpi resolution.
3.5.4. Detection of L -[ 35 S]-Methionine-Labeled Proteins
1. After silver staining and scanning, place gels on Whatman paper, cover with cello-
phane sheets, and dry on a vacuum dryer at 75 °C for at least 2-4 h ( see Note 11 ).
2. Expose the dried gels to “Storage Phosphor Screens” for2htoseveral days
(depending on signal intensity). Scan Storage Phosphor Screens with a Storm 840
Phosphor Imager at a resolution of 200 μm and a color depth of 16 bit (65536
gray scale levels) ( see Note 12 ).
3.6. Protein Identification by Mass Spectrometry (see Fig. 1)
1. For identification of proteins by MALDI-TOF MS, cut Coomassie-stained protein
spots from gels using a spot cutter (Proteome Work™) with a picker head of
2 mm and transfer into 96-well microtiter plates.
2. Digestion of proteins with trypsin and subsequent spotting of peptide solutions
onto the MALDI targets are performed automatically in the Ettan Spot Handling
Workstation (GE-Healthcare) using the following standard procedure (14) : wash
the gel pieces twice with 100 μL of 50 m M ammonium bicarbonate/50% (v/v)
methanol for 30 min and once with 100 μL of 75% (v/v) acetonitrile for 10 min.
After 17 min of drying, add 10 μL of trypsin solution containing 20 ng/μL trypsin
and incubate at 37 °C for 120 min. For peptide extraction, cover gel pieces with
60 μL of 50% (v/v) acetonitrile/0.1% (w/v) TFA and incubate for 30 min at 37 °C.
Transfer the supernatant containing peptides into a new microtiter plate and repeat
the extraction with 40 μL of the same solution. Dry the supernatants completely
at 40 °C for 220 min and dissolve the peptides in 2.2 μL of 0.5% (w/v) TFA/50%
(v/v) acetonitrile.
3. Spot 0.7 μL of this solution directly onto the MALDI target. Add 0.4 μL of matrix
solution and mix with the sample solution by aspirating the mixture five times.
Prior to the measurement in the MALDI-TOF instrument, dry the samples on the
target for 10-15 min.
4. Carry out MALDI-TOF MS analyses of spotted peptide solutions on a Proteome-
Analyzer 4700 (Applied Biosystems). The spectra are recorded in a reflector mode
in a mass range from 900 to 3700 Da. For one main spectrum, 25 subspectra with
100 spots per subspectrum are accumulated using a random search pattern. If the
autolytical fragment of trypsin with the monoisotopic (M+H) + m/z at 2211.104
reaches a signal-to-noise ratio (S/N) of at least 10, an internal calibration is
automatically performed using the peak for one point calibration. The peptide
search tolerance is 50 ppm, but the actual standard deviation is between 10 and
20 ppm.