A MALDI-TOF Mass Spectrometry Approach to Investigate the Defense Reactions
169
challenged Drosophila carrying a loss-of-function mutation of either the Toll
(ToW) or the imd gene are illustrated in Fig. 11.5. For the imd strain, all the DIMs
are induced after bacterial challenge (BC), with the exception of the two glycoforms of drosocin (DIMs 9 and 11), as expected, and the DIMs 15 and 16 (Fig.
11.5, top). Therefore, it seems that, like drosocin, the genes encoding DIMs 15
and 16 are under the control of the imd pathway. In the case of Drosophila carrying a Toll-mutation, only six molecules are induced after the infection: the two
glycoforms of drosocin (DIMs 9 and 11), metchnikowin (DIM 17), DIMs 15 and
16 and a molecule with a molecular mass of 4619 Da close to the molecular mass
of DIM 18 (4625 Da) (Fig. 11.5, bottom). Interestingly, this molecule at 4619 Da,
detected in the Toll-strain where no drosomycin is induced, has not been clearly
detected in the wild-type Drosophila strain because of the proximity of the drosomycin mass signal at m/z 4890.8. This confirms that suppression effects affect the
quality of the fingerprint of the Drosophila hemolymph. Furthermore, in double
mutants (imd/Tolt) no DIMs are detectable, suggesting that only the two distinct
pathways already reported (imd and Toll) control the gene expression of the
DIMs. We also analyzed the DIM expression in Drosophila mutants carrying a
dominant mutation of the Toll gene (Tow ob ; where the Toll receptor is constitutively activated; Lemaitre et al. 1996) and compared the mass spectra obtained
before and after immune challenge with the one recorded from wild-type flies
(data not shown). In unchallenged Toll gain-of-function mutants (Toll lOb ), most
of the DIMs are constitutively expressed confirming our first assumption that
DIMs are not products of bacterial origin (see above and for the mass spectra
Uttenweiler-Joseph et al. 1998).
In conclusion, all the unknown DIMs are controlled by the Toll pathway, with
the exception of DIMs 15-16 and DIM at 4619 Da which are controlled by the
imd pathway. In addition, all these experiments on Drosophila mutants gave valuable information on the place of the DIMs in the general scheme of Drosophila
immunity. However, the precise role of the DIMs remains to be clarified.
MALDI- TOP MS allows to follow, in a genetic model such as the fruitfly Drosophila melanogaster, the presence of molecules in different contexts of mutations
affecting a physiological process.
8
Identification of the DIMs by Sequencing
In an attempt to understand the precise function of the DIMs in the Drosophila
defense reactions, the induced molecules have to be structurally characterized.
On such minute amounts of molecules available from hemolymph samples,
methods for protein sequence analysis directly from complex mixtures can be
done by tandem MS (MS/MS) but only with MALDI ionization. One MS/MS
strategy involving ionization by MALDI is MALDI-Post Source Decay (PSD).
However limitations exist such as difficulties in interpreting a mass spectrum
resulting from this kind of fragmentation and also at the level of the mass precision for the selection of the ion for isolation, a prerequisite parameter in our
complex sample (for review, see Yates 1998). For this reason, we developed a
methodology based on the prepurification of the peptides by HPLC. However this
169
challenged Drosophila carrying a loss-of-function mutation of either the Toll
(ToW) or the imd gene are illustrated in Fig. 11.5. For the imd strain, all the DIMs
are induced after bacterial challenge (BC), with the exception of the two glycoforms of drosocin (DIMs 9 and 11), as expected, and the DIMs 15 and 16 (Fig.
11.5, top). Therefore, it seems that, like drosocin, the genes encoding DIMs 15
and 16 are under the control of the imd pathway. In the case of Drosophila carrying a Toll-mutation, only six molecules are induced after the infection: the two
glycoforms of drosocin (DIMs 9 and 11), metchnikowin (DIM 17), DIMs 15 and
16 and a molecule with a molecular mass of 4619 Da close to the molecular mass
of DIM 18 (4625 Da) (Fig. 11.5, bottom). Interestingly, this molecule at 4619 Da,
detected in the Toll-strain where no drosomycin is induced, has not been clearly
detected in the wild-type Drosophila strain because of the proximity of the drosomycin mass signal at m/z 4890.8. This confirms that suppression effects affect the
quality of the fingerprint of the Drosophila hemolymph. Furthermore, in double
mutants (imd/Tolt) no DIMs are detectable, suggesting that only the two distinct
pathways already reported (imd and Toll) control the gene expression of the
DIMs. We also analyzed the DIM expression in Drosophila mutants carrying a
dominant mutation of the Toll gene (Tow ob ; where the Toll receptor is constitutively activated; Lemaitre et al. 1996) and compared the mass spectra obtained
before and after immune challenge with the one recorded from wild-type flies
(data not shown). In unchallenged Toll gain-of-function mutants (Toll lOb ), most
of the DIMs are constitutively expressed confirming our first assumption that
DIMs are not products of bacterial origin (see above and for the mass spectra
Uttenweiler-Joseph et al. 1998).
In conclusion, all the unknown DIMs are controlled by the Toll pathway, with
the exception of DIMs 15-16 and DIM at 4619 Da which are controlled by the
imd pathway. In addition, all these experiments on Drosophila mutants gave valuable information on the place of the DIMs in the general scheme of Drosophila
immunity. However, the precise role of the DIMs remains to be clarified.
MALDI- TOP MS allows to follow, in a genetic model such as the fruitfly Drosophila melanogaster, the presence of molecules in different contexts of mutations
affecting a physiological process.
8
Identification of the DIMs by Sequencing
In an attempt to understand the precise function of the DIMs in the Drosophila
defense reactions, the induced molecules have to be structurally characterized.
On such minute amounts of molecules available from hemolymph samples,
methods for protein sequence analysis directly from complex mixtures can be
done by tandem MS (MS/MS) but only with MALDI ionization. One MS/MS
strategy involving ionization by MALDI is MALDI-Post Source Decay (PSD).
However limitations exist such as difficulties in interpreting a mass spectrum
resulting from this kind of fragmentation and also at the level of the mass precision for the selection of the ion for isolation, a prerequisite parameter in our
complex sample (for review, see Yates 1998). For this reason, we developed a
methodology based on the prepurification of the peptides by HPLC. However this