Making a Protein Extract from Plant Pathogenic Fungi for Gel- and LC-Based Proteomics - Plant Proteomics: Methods and Protocols - page 98

Before the second dimension, IPG strips are equilibrated fol-
lowing two steps. Firstly, it is carried out with 2 % (w/v) DTT in
equilibration buffer for 10 min in agitation at room temperature;
secondly, it is done with 2.5 % (w/v) iodoacetamide in equilibra-
tion buffer for 10 min in agitation at room temperature.
The second dimension is performed in the same way explained
in Subheading 3.3 , but using Criterion Stain-Free precast Gels,
Tris-HCl, and 8-16 % linear gradient IPG strips (Bio-Rad). After
protein staining, spots can be analyzed using the PD-Quest soft-
ware (Bio-Rad).

Protein sample (obtained according to Subheading 3.2 ) is digested
at 37 °C overnight using trypsin. Tryptic peptides are cleaned and
concentrated in a C18 Cartridges Octadecyl C18/18 % (Applied
Separations), dried in a Speed-vac ® Concentrator, and re-suspended
in 50

3.6 Protein
Separation by LC

L of a 5 % ACN and 0.1 % formic acid solution. Finally,
peptides are injected to the LC system (i.e., a Finnigan Surveyor an
HPLC system). All these processes were made according to the
Proteomic Service protocols of SCAI, University of Córdoba.

μ

3.7 Protein
Identifi cation

The bands or spots are cut out and digested with trypsin. Tryptic
peptides are analyzed in a mass-spectrometer (i.e., a 4800
Proteomics Analyzer MALDI-TOF/TOF, Applied Biosystems).
In this case, the eight most abundant peptide ions are chosen for
an MS/MS analysis. A PMF search and a combined search (+MS/
MS) are performed using the nrNCBI database of proteins with
the MASCOT algorithm ( www.matrixscience.com ) . All these pro-
cesses were made according to Proteomic Service protocols of
SCAI, University of Córdoba ( see Note 12 ).

3.7.1 Gel-Based
Techniques

Peptides are detected for example in an LTQ-Orbitrap equipped
with a nanoelectrospray ion source (nESI). The acquired data can
be analyzed with Proteome Discoverer v1.3 software (Thermo
Fisher Scientifi c, USA) and MASCOT ( http://www.matrix-
science.com ) or SEQUEST ( http://fi elds.scripps.edu/sequest/ )
algorithms, using the public Botrytis cinerea Database from Botrytis
cinerea Sequencing Project of Broad Institute of Harvard and MIT
( http://www.broadinstitute.org/annotation/genome/botrytis_
cinerea/MultiHome.html ) , according to Proteomic Service proto-
cols of SCAI, University of Córdoba ( see Note 13 ).

3.7.2 Gel-Free
Techniques

4 Final Remarks

With the use of gel-based techniques, around 50 bands and 500
spots were resolved subjected to 1- and 2-DE, respectively, from B.
cinerea B05.10 mycelia protein extracts [ 45 ]. With the use of
label-free LC-based approach, around 240 protein species were