Biology Reference
In-Depth Information
10. The data acquired on the mass spectrometer are used for iden-
tifi cation searches in a protein databases. Several types of soft-
ware may be used for automated and manual interpretation of
the results. A non-exhaustive list of the Web sites offering this
feature is given here:
(a)
http://fasta.bioch.virginia.edu/fasta_www2/fasta_list2.
shtml
(b)
http://www.matrixscience.com/search_form_select.html
(c)
http://prospector.ucsf.edu/
(d)
http://www.expasy.org/tools/
The MS spectrum provides a list of masses corresponding to
peptides generated during the digestion of the protein. The
MS/MS spectra allow the sequence of amino acids from a spe-
cifi c peptide selected for fragmentation. The obtained masses
and sequences are compared with existing databases. After
identifi cation, literature survey is usually requested to link the
results obtained to the initial biological question. More infor-
mation about MS can be found in the following selection of
excellent reviews: general mass spectrometry [
17
] in model
plants [
18
,
19
] and in non-model plants [
20
].
Finally, these steps are given as example of the protocols that
are working well in our laboratory, and are used frequently on dif-
ferent matrices: potato, poplar, alfalfa, oak, alder, and other plants,
but also on mosses, fungi, etc. The number of spots depends
greatly on the tissue used, the explored pI range, whether pre-
fractionation steps are used, and so on. Usually, we consider that a
good result is between 70 and 95 % of identifi ed proteins, varying
according to the species, and the availability of its genome.
4
Notes
1. Urea and thiourea help the solubilization of proteins.
2. DTT preserves the fully reduced state of denatured, unalkyl-
ated proteins.
3. The classical colorimetric method of Bradford cannot be used
for protein quantifi cation, as the components of the labelling
solution interfere with the Bradford solution (e.g., the high
concentration of urea, detergent).
4. The DMF solution should not be contaminated with water,
which degrades DMF to amine compounds. Select a DMF
solution of 0.005 % H
2
O, 99.8 % of purity, and this solution
should be replaced at least every 3 months.
5. A dye swap is always recommended to avoid preferential
labelling.
