Biology Reference
In-Depth Information
3.7. Protein
Identifi cation
by MALDI-TOF MS
and Data Analysis
1.
0.5 mL of tryptic digest is mixed with 1 mL of saturated aqueous
2,5-dihydroxybenzoic acid matrix solution and spotted onto a
target plate and dried.
2.
Mass spectra are acquired on an Ultrafl ex TOF/TOF mass
spectrometer in the refl ector mode. The spectrometer is cali-
brated using the peptide external calibration standard, and
internal calibration is carried out using trypsin autolysis peaks
at
m/z
842.51 and
m/z
2,211.10.
3.
Peaks in the mass range of
m/z
500-5,000 are used to generate
a peptide mass fi ngerprint that is searched against the updated
NCBInr database using Mascot Peptide Mass Fingerprint
software (Matrix Science, London, UK;
http://www.matrixscience.
com/search_form_select.html
). The following parameters are
used for the search:
Homo sapiens
(or relevant taxonomy);
tryptic digest with a maximum of one missed cleavage; carb-
amidomethylation of cysteine; protein N-terminal acetylation,
methionine oxidation, and glutamine to pyroglutamate as
variable modifi cation; and a mass tolerance of ±50 ppm (see
Note 14).
4.
A positive identifi cation is accepted based on a signifi cant
Mascot MOWSE score (
p
< 0.05), at least 6 peptide masses
matching a particular protein, matched peptides covering
>20% of the matched protein sequence and general agreement
between the observed and theoretical molecular weight on
2D gels.
3.8. Validation
by Immunoblotting
1.
Separate samples by 1D SDS-PAGE and transfer electropho-
retically to PVDF membrane using transfer buffer in a transfer
tank (Bio-Rad Transfer-Blot cell). Wet the PVDF membrane in
100% methanol for 1 min, then in transfer buffer for 5 min and
place on top of the gel without air bubbles. Sandwich mem-
brane and gel between two pieces of Whatman paper soaked in
transfer buffer. Remove air bubbles with a plastic pipette and
fi x fi rmly in the tank cassette. Insert the cassette into the transfer
tank containing transfer buffer and make sure the membrane is
located between the gel and the anode. Connect the tank to an
appropriate power supply and transfer at 350 mA for 5 h.
2.
Once transfer is complete, remove the “sandwich” from the
cassette and disassemble. The colored molecular weight
markers should be clearly visible on the membrane if the
transfer has worked.
3.
Incubate the membrane in 500 mL of 5%low-fat milk in TBS-T
for 1 h at room temperature on a rocking platform. The mem-
brane can also be left in blocking buffer over night at 4°C.
4.
Discard the blocking buffer and rinse the membrane with
TBS-T prior to the addition of a dilution of primary antibody.
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