Biology Reference
In-Depth Information
(this technique combines a size-based protein separation with an
in-gel digestion of the resulting fractions) [
51
]. This GeLC-MS/
MS strategy paves the way towards the analysis on large-scale fun-
gal response environmental cues on the basis of quantitative shot-
gun protein-profi ling experiments. The case of multidimensional
protein identifi cation technology (MudPIT), which allows the
identifi cation of a much larger number of proteins compared to
gel-based methods, has been a drawback due to the lack of quanti-
tative data [
52
,
53
]. MudPIT was used to analyze germling growth
mechanisms in
Uromyces appendiculatus
by comparing germinat-
ing asexual uredospores to inactive spores [
54
].
MS is the basic technique for global proteomic analysis due to
its accuracy, resolution, and sensitivity (in the femtomole to atto-
mole concentration range), and due to the fact that it has the
capacity for a high throughput. Not only does it allow profi ling a
proteome, but also and more important, it allows the identifi cation
of protein species and the characterization of posttranslational
modifi cations and interactions. Proteins are identifi ed from mass
spectra of intact proteins (top-down proteomics), or peptide frag-
ments obtained after enzymatic (mostly digested with trypsin) or
chemical treatment (bottom-up proteomics). Protein species are
identifi ed by comparison of the experimental spectra, while the
theoretical ones were obtained
in silico
from protein, genomic,
EST sequence, or MS spectra databases. For this purpose, different
instrumentation, algorithms, databases, and repositories are avail-
able [
55
,
56
].
Although 2-DE remains as a standard tool for fungal pro-
teomic research, current efforts are focussed on alternative gel-free
shotgun strategies to identify and quantify proteins. The coupling
of nanoscale separations (nanocapillary or nLC) with automated
MS/MS has enhanced the development of this methodology.
Using LC MS/MS, the complex mixtures of proteins are digested
to peptides (normally with trypsin), which are separated according
to their hydrophobicity by nLC, and then the eluted peptides are
introduced into the mass spectrometer [
57
]. For example, a gel-
free analysis from mycelium and secreted proteins of
B. cinerea
B05.10 and T4 strains has been carried out using a SYNAPT
HDMS mass spectrometer (Waters) interfaced with a NanoAcquity
UPLC System (Waters) [
45
,
59
,
60
]. A total of 197 and 73 pro-
teins were identifi ed from mycelia and secreted proteins, respec-
tively (Fig.
3
). Recent reviews study techniques, software, and
statistical analyses used in gel-free quantitative proteomics, and
discuss about its strengths and limitations [
3
,
61
,
62
].
Although several methods for proteomic analysis of limited
fungal species have been published [
4
,
12
,
63
,
64
], procedures for
protein extraction as well as gel-based and gel-free analysis condi-
tions are progressively evolving to study individual characteristics
of fungal species.
