Biology Reference
In-Depth Information
The fi rst steps prior to MS analysis (biological system dependent),
although not always realized, are key to the success of the experiment,
despite the fact we use to consider them as trivial. An adequate and
correct experimental design, together with the statistical analysis of
the data, is requested. Surprisingly, this tends to be ignored or not
properly considered, even in current literature (
see
Chapter
5
)
.
Only when a protein is extracted, solubilized, and visualized,
it can be identifi ed and quantifi ed. For this reason, attention must
be paid to procedures for protein extraction, especially in the case
of recalcitrant proteins (highly hydrophobic, with extreme pI or
Mr
) or plant tissues rich in nonprotein material, such as phenolics,
lipids, salts, and polysaccharides (
see
Chapters
7
,
8
,
33
). Apart
from this, it will be easy for most biochemists to understand and
perform by themselves these preliminary steps, while mass spec-
trometry and data analysis (identifi cation, quantifi cation, and
characterization) require a real, deep expertise. This, together
with the cost of the mass spectrometers and associated bioinfor-
matic packages, justifi es the use of proteomics services. It would
be impossible, however, to exploit the huge amount of informa-
tion generated by a mass spectrometer having no knowledge of
the experimental system, the protein extract and solution, and of
how the last was obtained from the previous.
Despite the technological achievements in proteomics, only a
tiny fraction of the cell proteome has been characterized so far, and
only for a few biological systems (human, fruit fl y, Arabidopsis, rice,
Chlamydomonas). Even for these organisms, the function of quite
a number of proteins remains to be investigated. Proteomics tech-
niques have a number of limitations, such as sensitivity, resolu-
tion, and speed of data capture. They also face a number of
challenges, such as deeper proteome coverage, proteomics of
unsequenced “orphan” organisms, top-down proteomics, protein
quantifi cation, PTMs or interactomics, among others. Most of
these limitations and challenges refl ect the diffi culty of working
with the biological diversity of proteins and their range of physico-
chemical properties (
see
Chapter
27
)
. Compared to other biological
systems, plants present a number of characteristics that makes dif-
fi cult the obtention of a good protein extract (
see
Chapter
9
)
. On
the other side, plants share with the other systems the high protein
dynamic range that makes impossible to detect minor proteins. To
face this problem, a number of approaches may be proposed,
including subcellular or protein fractionation (
see
Chapters
10
,
26
,
27
,
31
-
36
,
38
), protein depletion by using antibodies, or the most
recent technique developed: the combinatorial peptide ligand
libraries or CPLL, described in detail by its inventor Prof. Righetti
in Chapter
9
.
For specifi c areas, like subcellular proteomics, diffi cul-
ties are associated with the isolation of relatively pure samples from
plant material free of contamination (
see
Chapters
31
-
36
)
. In this
regard, proteomics has shown us that the static view of proteins