Biology Reference
In-Depth Information
3.3. Preparation
of Peptides for MALDI
Mass Spectrometry
1.
Add 5
L of dissolving solution into each digest-containing
sample well (or tube).
μ
2.
Centrifuge for 1 min at 2,000 rpm.
3.
Vortex the plate for 10 min at shaking position (or vortex tubes
for 1 min).
3.3.1. Peptide
Reconstitution
4.
Centrifuge again for 1 min at 2,000 rpm.
Dried-droplet technique
3.3.2. Spotting MALDI
Samples on a Plate
( see Note 9)
1.
Spot 0.5-0.8
L of sample solution on the center of a MALDI
target spot. Discard the pipette tip.
μ
2.
Spot immediately 0.3-0.4
L of matrix solution on the sample
drop and mix both solutions pipetting 3-4 times if needed.
Discard the pipette tip.
μ
3.
Repeat steps 1 and 2 for all samples.
4.
Dry the spotted MALDI plate at room temperature.
3.4. MALDI Mass
Spectrometry
MS spectra of protein digests are typically collected in the refl ector
positive ion mode with a mass range of 700-4,000 Da and 50 laser
shots per desorption spot (1,250 shots per spectrum). The peptide
MS peaks with a signal-to-noise ratio above 20 are selected for
MS/MS analysis. A maximum of 45 MS/MS spectra are collected
per MALDI sample spot. The precursor mass window is 200 relative
resolution (fwhm). Calibration is performed internally using com-
mon trypsin autolysis peaks or externally using a standard peptide
calibration mixture before each series of MS or MS/MS experiments
(see Note 10).
3.4.1. Mass Spectrometry
with the 4800 MALDI-TOF/
TOF Proteomics Analyzer
MS data search of all the spectra against the taxonomy of interest
is performed using the NCBInr database, with the GPS Explorer™
Software v3.6 and Mascot search engine or with the Protein Pilot
(vs. 3.0) software. Mass tolerance is usually set at 50-100 ppm for
precursor ions and 0.5 Da for fragment ions. In Mascot usually up
to two missed cleavages are declared, and oxidation (M) and acety-
lation (K) are declared as variable modifi cations. Cysteine alkyla-
tion is routinely performed for 2D gel analysis and hence should be
declared as fi xed modifi cation (see Note 11).
3.4.2. MS Data Analysis
2D DIGE experiments are often based on the “minimal” labeling
method, in which around 5% of total protein is covalently labeled
((
1
) and Chapter
3.5. Examples
3.5.1. Sensitivity
of Detection
10). For the experiment described below, the
method was slightly changed by not adding lysine to the reaction
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