CHAPTER 11
A MALDI-TOF Mass Spectrometry Approach
to Investigate the Defense Reactions
in Drosophila melanogaster, an Insect Model
for the Study of Innate Immunity
PH. BULET I and S. UTTENWEILER-JOSEPH 2
1
Summary
A protocol consisting of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), high performance liquid chromatography (HPLC),
Edman degradation and cDNA cloning has been optimized for the rapid identification and structural characterization of peptides induced during the humoral
immune response of Drosophila. MALDI-MS analysis led to the detection of at
least 24 immune-induced molecules (DIMs) in the hemolymph (body fluid, 0.1
!ll) of a single fruitfly in response to an experimental challenge. The use of micropurification by HPLC, peptide sequencing and molecular biology techniques
allowed the characterization of three out of the 24 DIMs from a hemolymph sample collected from less than 140 flies. This study clearly shows that MALDI-MS
differential mapping constitutes a powerful tool to detect compounds involved in
inducible physiological processes.
2
Introduction
The present interest for insect immunity was stimulated by the recent discovery
of striking parallels that exist between insect immunity and innate immunity in
vertebrates (Ezekowitz and Hoffmann 1998). Similarly, studies on vertebrate
innate immunity have been encouraged by the perception that it plays a role in
the induction of adaptive immunity and it directs particular effector mechanisms
in this response (for review, see Fearon and Locksley 1996). The discovery of similarities between vertebrate and insect innate immunity suggests that Drosophila
melanogaster, through its powerful genetics, is a suitable model for evolutionary
studies on innate immunity (for review, see Meister et al. 1997).
The immunity of Drosophila, like that of other insects, relies on both cellular
and humoral reactions. Cellular defense includes the phagocytosis and encapsulation of intruders by blood cells. This aspect has mostly been described at the
I Institut de Biologie Moleculaire et Cellulaire, Unite Propre de Recherche 9022 du Centre National de
la Recherche Scientifique, 15, rue Rene Descartes, 67084 Strasbourg Cedex, France.
2 Present address: European Molecular Biology Laboratory (EMBL), Protein & Peptide Group,
Meyerhofstrasse, 1,69117 Heidelberg, Germany.
A MALDI-TOF Mass Spectrometry Approach
to Investigate the Defense Reactions
in Drosophila melanogaster, an Insect Model
for the Study of Innate Immunity
PH. BULET I and S. UTTENWEILER-JOSEPH 2
1
Summary
A protocol consisting of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), high performance liquid chromatography (HPLC),
Edman degradation and cDNA cloning has been optimized for the rapid identification and structural characterization of peptides induced during the humoral
immune response of Drosophila. MALDI-MS analysis led to the detection of at
least 24 immune-induced molecules (DIMs) in the hemolymph (body fluid, 0.1
!ll) of a single fruitfly in response to an experimental challenge. The use of micropurification by HPLC, peptide sequencing and molecular biology techniques
allowed the characterization of three out of the 24 DIMs from a hemolymph sample collected from less than 140 flies. This study clearly shows that MALDI-MS
differential mapping constitutes a powerful tool to detect compounds involved in
inducible physiological processes.
2
Introduction
The present interest for insect immunity was stimulated by the recent discovery
of striking parallels that exist between insect immunity and innate immunity in
vertebrates (Ezekowitz and Hoffmann 1998). Similarly, studies on vertebrate
innate immunity have been encouraged by the perception that it plays a role in
the induction of adaptive immunity and it directs particular effector mechanisms
in this response (for review, see Fearon and Locksley 1996). The discovery of similarities between vertebrate and insect innate immunity suggests that Drosophila
melanogaster, through its powerful genetics, is a suitable model for evolutionary
studies on innate immunity (for review, see Meister et al. 1997).
The immunity of Drosophila, like that of other insects, relies on both cellular
and humoral reactions. Cellular defense includes the phagocytosis and encapsulation of intruders by blood cells. This aspect has mostly been described at the
I Institut de Biologie Moleculaire et Cellulaire, Unite Propre de Recherche 9022 du Centre National de
la Recherche Scientifique, 15, rue Rene Descartes, 67084 Strasbourg Cedex, France.
2 Present address: European Molecular Biology Laboratory (EMBL), Protein & Peptide Group,
Meyerhofstrasse, 1,69117 Heidelberg, Germany.