172
PH. BULET and S. UTTENWEILER-JOSEPH
The MALDI mass spectra obtained on GFP+ and GFP- pieces of tracheal tissue
were relatively simple and quite similar, with the exception of a peak at m/z 4910
only detectable in the mass spectrum from fluorescent trachea (Fig. 11.6). It
should be noticed that these mass spectra were acquired at high laser power, that
can result in (i) a loss of resolution and (ii) a shift of the measured mass to a
higher mass than expected. The peak detected at m/z 4910 in the mass spectrum
of GFP+ trachea corresponds to endogenous drosomycin as controlled with
recombinant drosomycin in the same conditions of analysis. This observation
strongly suggests that the respiratory tract of Drosophila is not only acting as a
physical barrier but as the potency to develop a local immune response in order
to fight off a natural microorganism penetration.
The detection of an already characterized protein in a biological tissue can be
rapidly and easily performed by MALDI- TOP MS without the need of any purification step. This technology can be applied to all biological models as already shown
in other publications (see for example references in the Introduction).
10
Acknowledgments
The authors are indebted to Dr. Jules A. Hoffmann for his continuous interest in
this study and to Dr. Alain Van Dorsselaer for providing the facilities in mass
spectrometry.
11
Conclusion
The data obtained by differential MALDI-TOF MS analysis of crude hemolymph
from bacteria-challenged and control Drosophila illustrate the great potential of
this method for the detection of molecules induced in a complex biological process. However, this MALDI-TOF MS approach appeared to be more appropriate
for the analysis of low molecular mass peptides. For the complementary analysis
of higher molecular mass proteins, 2-D gel electrophoresis associated to mass
spectrometry techniques is certainly more adapted.
References
Ashida M, Brey P (1997) Recent advances in research on the insect prophenoloxidase cascade. In
Molecular mechanisms of immune responses in insects, P. T. Brey and D. Hultmark, eds.: Chapman
& Hall: pp 135-172
Beavis R, Chait B (1990) Rapid, sensitive analysis of protein mixtures by mass spectrometry. Proc Nat!
Acad Sci USA 87: 6873-6877
Beavis R, Chait B (1996) Matrix-assisted laser desorption ionization mass spectrometry of proteins.
Methods in Enzymol270: 519-551
Braun A, Lemaitre B, Lanot R, Zachary D, Meister M (1997) Drosophila immunity: analysis oflarval
hemocytes by P-element-mediated enhancer trap. Genetics 147: 623-634
Bulet P, Dimarcq J, Hetru C, Lagueux M, Charlet M, Hegy G, Van Dorsselaer A, Hoffmann J (1993) A
novel inducible antibacterial peptide of Drosophila carries an O-glycosylated substitution. J Bioi
Chern 268: 14893-14897
Burlingame P, Boyd R K, Gaskell S J (1998) Mass Spectrometry. Anal Chern 70: 647R-716R
PH. BULET and S. UTTENWEILER-JOSEPH
The MALDI mass spectra obtained on GFP+ and GFP- pieces of tracheal tissue
were relatively simple and quite similar, with the exception of a peak at m/z 4910
only detectable in the mass spectrum from fluorescent trachea (Fig. 11.6). It
should be noticed that these mass spectra were acquired at high laser power, that
can result in (i) a loss of resolution and (ii) a shift of the measured mass to a
higher mass than expected. The peak detected at m/z 4910 in the mass spectrum
of GFP+ trachea corresponds to endogenous drosomycin as controlled with
recombinant drosomycin in the same conditions of analysis. This observation
strongly suggests that the respiratory tract of Drosophila is not only acting as a
physical barrier but as the potency to develop a local immune response in order
to fight off a natural microorganism penetration.
The detection of an already characterized protein in a biological tissue can be
rapidly and easily performed by MALDI- TOP MS without the need of any purification step. This technology can be applied to all biological models as already shown
in other publications (see for example references in the Introduction).
10
Acknowledgments
The authors are indebted to Dr. Jules A. Hoffmann for his continuous interest in
this study and to Dr. Alain Van Dorsselaer for providing the facilities in mass
spectrometry.
11
Conclusion
The data obtained by differential MALDI-TOF MS analysis of crude hemolymph
from bacteria-challenged and control Drosophila illustrate the great potential of
this method for the detection of molecules induced in a complex biological process. However, this MALDI-TOF MS approach appeared to be more appropriate
for the analysis of low molecular mass peptides. For the complementary analysis
of higher molecular mass proteins, 2-D gel electrophoresis associated to mass
spectrometry techniques is certainly more adapted.
References
Ashida M, Brey P (1997) Recent advances in research on the insect prophenoloxidase cascade. In
Molecular mechanisms of immune responses in insects, P. T. Brey and D. Hultmark, eds.: Chapman
& Hall: pp 135-172
Beavis R, Chait B (1990) Rapid, sensitive analysis of protein mixtures by mass spectrometry. Proc Nat!
Acad Sci USA 87: 6873-6877
Beavis R, Chait B (1996) Matrix-assisted laser desorption ionization mass spectrometry of proteins.
Methods in Enzymol270: 519-551
Braun A, Lemaitre B, Lanot R, Zachary D, Meister M (1997) Drosophila immunity: analysis oflarval
hemocytes by P-element-mediated enhancer trap. Genetics 147: 623-634
Bulet P, Dimarcq J, Hetru C, Lagueux M, Charlet M, Hegy G, Van Dorsselaer A, Hoffmann J (1993) A
novel inducible antibacterial peptide of Drosophila carries an O-glycosylated substitution. J Bioi
Chern 268: 14893-14897
Burlingame P, Boyd R K, Gaskell S J (1998) Mass Spectrometry. Anal Chern 70: 647R-716R