Biology Reference
In-Depth Information
been used to identify several posttranslational modifi cations, such as
citrullinated proteins[
40
], carbonylated proteins [
41
], proteins
containing nitrotyrosine [
42
,
43
] or hydroxynonenal adducts [
44
,
45
].
Although very successful, this strategy has a major caveat, i.e.
the fact that a minor and modifi ed spot detected with the antibody
may comigrate with a major, unrelated spot which will be the one
identifi ed by the downstream mass spectrometry process, resulting
in a major mistake. The severity of this problem increases with spot
crowding and thus with the complexity of the biological extract.
There is however an easy counter, at least for acidic and neutral
proteins, which is the use of narrow range pH gradients that greatly
decrease the probability of comigration. Such narrow pH range
gels have been used under a variety of circumstances [
46
-
49
], and
have proven very effi cient, especially when used in conjunction
with prefractionation techniques [
50
].
5
Analysis of Intact Proteins and of Posttranslational Modifi cations
The more proteomics progresses, the more it stresses the impor-
tance of posttranslational modifi cations in biology (e.g. in [
51
,
52
]). However, not all posttranslational modifi cations can be easily
studied through the use of an affi nity reagent, and some, as meth-
ylations, are not easily detected except on abundant proteins [
53
].
For the study of these diffi cult, and sometimes unknown,
modifi cations, 2D gel-based proteomics is a platform of choice,
either through selective and transient labeling [
54
], or by using the
fact that many modifi cations induce a pH shift and thus a separa-
tion of the modifi ed protein from the bulk of the unmodifi ed one.
This can be used in the fi eld of protein cleavage [
55
,
56
], but also
as a preparative tool to isolate the modifi ed spot, thereby leading
to an easier identifi cation of the modifi cations. Such an approach
has been used for the identifi cation of thiol oxidations [
57
-
59
].
More generally, the separating ability of 2D gels, coupled with
their ability to be used as a micropreparative tool [
60
], now allow
to set very comprehensive maps of protein modifi cations [
61
,
62
].
In this area, the use of ultra narrow pH gradient [
47
], with their
almost infi nite resolving power [
63
,
64
] should be extremely use-
ful, by providing a very thorough deconvolution of the protein
spots into individual protein species.
6
Conclusions
Nowadays, 2D gel-based proteomics is usually selected on the basis
of its robustness and relatively low cost, among the proteomic
techniques. It can be reasonably anticipated, however, that the uses
directed toward the identifi cation of posttranslational modifi cations,
