Biology Reference
In-Depth Information
cases, the protein subclasses are defi ned by a common biochemical
feature, and quite often this biochemical feature is a posttranslational
modifi cation, for which an affi nity reagent exists.
When such an affi nity reagent exists, two types of strategies can
be devised to perform proteomic studies targeted for the proteins
recognized by the affi nity reagent.
In the fi rst strategy, the affi nity reagent is used as a pre-proteomic
preparative tool, aiming at selecting the proteins or peptides of
interest in the complex starting extract. The selected proteins/
peptides are then analyzed by a classical proteomic setup.
This strategy is the only one that can be used with shotgun pro-
teomics, but it can also be used with 2D gel-based proteomics as well.
In the second strategy, the affi nity reagent is used as an analyti-
cal reagent, after the separation stage in the proteomic setup, in
order to detect selectively the proteins of interest. This strategy
works extremely well with 2D gel-based proteomics, thanks to the
easy interface provided by the blotting process between 2D gels
and affi nity reagents.
Thus, the fi nal performance of the whole process will largely
depend on the effi ciency of the affi nity reagent under the prepara-
tive and analytical setups, and it must be kept in mind that for
several reasons, the affi nity reagent is always used in a large excess
over the biological extract.
Then two major cases can be distinguished:
1. The affi nity reagent is not a protein
Typical examples of that confi guration are represented by
boronic acid for sugars and either IMAC or metal oxides for
phosphate. These nonproteinaceous reagents are best used in
the preparative mode, and work wonderfully with shotgun pro-
teomics [
34
].
2. The affi nity reagent is a protein
In this case, the suitability of the reagent as a preparative tool
will depend not only on its selectivity, but also on the existence
of mild elution conditions that are able to elute all the analytes
without eluting out the affi nity reagent.
Lectins represent such a happy case and have been used as a
preparative reagent in 2D gel-based proteomics [
35
] and in shot-
gun proteomics [
36
-
38
].
They have also been used as a post-2D gel selective detection
reagent [
39
].
Conversely, antibodies represent a case where elution without
polluting the extract with antibodies is very diffi cult. They are there-
fore much better used as analytical reagents on blots rather than pre-
parative reagents. Thus, 2D gel-based proteomics is a very effi cient
strategy to use in conjunction with antibodies, and this strategy has
