Biology Reference
In-Depth Information
9. Automatically statistical analysis is followed within SameSpots
software. A principal component analysis (PCA) [
40
], correla-
tion analysis (CA), and power analysis are implemented in
SameSpots which allow studying the protein abundance trends
in the experiment (Fig.
2b-d
).
10. Choose a picking image for further analysis by mass
spectrometry.
Mass spectrometry analysis in the case of protein identifi cation
from spots is usually performed by a sequential strategy which con-
sists in the identifi cation of peptides obtained after in-gel digestion
by peptide mass fi ngerprint (PMF) in a MALDI-TOF instrument
followed by identifi cation of those unidentifi ed spots by tandem
mass spectrometry (MS/MS) in an LC-MS/MS instrument. In
the case of samples containing complex protein mixtures, the iden-
tifi cation of proteins is directly performed after in-solution protein
digestion followed by LC-MS/MS.
3.12 Protein
Identifi cation and
Database Searches
1.
MALDI-TOF mass spectrometry
. Spots or bands of interest are
excised with the aid of a spot picker. The peptides for mass
spectrometry analysis are obtained after in-gel digestion [
33
].
The tryptic fragments are desalted using Zip Tips C18 accord-
ing to the manufacturer's instructions. Eluted peptides are
completely dehydrated in a vacuum centrifuge and resus-
pended in 0.1 % TFA (
see
Note 29
). A droplet of 0.5
μ
L is
spotted onto the AnchorChip MALDI target. After add 0.5
L of
the CHCA matrix solution to the droplets and allow to air-dry
at room temperature (
see
Note 30
). External mass calibration
was performed using a peptide calibration mixture at 100 fmol/
μ
μ
L is spotted onto the
AnchorChip MALDI target. After add 0.5
L concentration. A droplet of 0.5
μ
L of the CHCA
matrix solution to the droplets and allow to air-dry at room
temperature (
see
Note 31
). Delayed-extraction MALDI-TOF
spectra are recorded using an Autofl ex (Bruker Daltonics)
equipped with a nitrogen laser. Spectra were obtained in the
positive-ion refl ection mode.
μ
60 laser shots are acquired
within each spot after manual identifi cation of areas with strong
signals. Summed spectral signal acquired in each spot is stored
and used for further database searches and subsequent protein
identifi cation (
see
Note 32
).
Protein identifi cation is performed by peptide mass map-
ping and database searching using the in-house licensed search
engine MASCOT (Matrix science). The following parameters
are set for searches using MASCOT: enzyme: trypsin; fi xed
modifi cations: carbamidomethyl cysteine; variable modifi ca-
tions: oxidation of methionine; peptide tolerance: ±0.1 Da;
peptide charge: 1H
+
monoisotopic; number of missed cleavage
sites: up to 1 missed cleavage site; decoy database searches
≥
