4 - Central Institute of Brackishwater Aquaculture

National Workshop-cum-Train~ng on Bloinformatlcs and Information Management in Aquaculture
At the protein level, the method used today is still two-dimensional (2D) gel
electrophoresis. This separates the denatured proteins according to two
independent criteria, the isoelectric point in the first dimension
(isoelectrofocusing) and the apparent molecular mass in the second one
(electrophoresis in the presence of SDS). On the 2 D gels obtained, several
hundreds to several thousands of protein spots (i.e. of gene products) are
revealed. These proteins can then be identified or characterized by different
methods. Immunological characterization can be done when the identification of
a spot is a priori suspected or when we are looking for an already known protein
on a 2 D gel. The classical protein sequencing of the N-terminal first 10 or 15
amino acids, or of internal sequences, is a very reliable but expensive and
relatively slow running method (one spot a day) and requires reasonable
amounts of protein. The combination of different criteria can be adopted to
identify a protein spot faster and at a lower cost: It permits one to
unambiguously identify a protein, if its gene is already present in the databanks.
More samples can be analysed for a lower cost than with Edman sequencing.
However, protein identifications are performed today by mass spectrometry. The
MALDI-TOF (Matrix Assisted Laser Desorption Ionisation - Time of Flight) mass
spectrometers give masses of peptides obtained after trypsin digestion of the
spots. These high-throughput apparatuses permit a fast identification of
numerous proteins when detailed genomic information is available on the studied
species. The data obtained ('peptide mass fingerprints') are compared to those
generated from the databanks such as SwissProt or 'rEMBL. Another type of
mass spectrometer, the ESI-MS/MS (Electro Spray Ionisation - Mass
Spectrometer / Mass Spectrometer), gives, by fragmentation of the trypsic
peptides, values that are diagnostic of amino acid sequences. Then, as with
Edman sequencing, the sequence itself can be compared to homologous ones
from other species and is thus preferred when the genome of the studied species
is not well represented in databanks.
Internet resource:
http://www.ebi.ac.uk/2can/databases/protein.html
http://www.expasy.ch/enzyme/
5. Metabolomics
Metabolomics is a powerful emerging technology, whereby the total metabolite
composition (the metabolome) of an organism is analyzed. By characterizing the
metabolome of an organism at different developmental stages, or following
exposure to different conditions, global shifts in its metabolism can be followed.
This approach complements studies in which changes in the transcriptome and
proteome are monitored. By combining global metabolomic analysis with
transcriptomics and proteomics it is possible, for the first time, to gain a holistic
view of the complex interactions between genes and metabolites. New
metabolomic technologies will lead to a greater understanding of metabolism
than was possible using previous profiling approaches limited to particular
classes of compounds. In addition, metabolomics offers a powerful technology to
characterise genes of unknown function, whereby the expression of the gene is
manipulated by mutagenesis or genetic engineering and its identity inferred from
the resulting metabolic changes. In particular, studies of plants and microbes are
important in identifying molecular determinants of food quality, safety and
nutrition, and opportunities for the exploitation of novel metabolic products. Plant
Molecular Biology (PMB), Biochemistry of Metabolic Regulation in Plants