A MALDI-TOF Mass Spectrometry Approach to Investigate the Defense Reactions
163
the 24 induced molecules do not result from bacteria. This result was confirmed
by the analysis of Drosophila mutants (see below).
In summary, none ofthe 24 induced molecules detected by MALDI-TOF MS in
Drosophila hemolymph are of bacterial origin or coming from the lysis of the
Drosophila blood cells. Therefore they represent molecules involved in the systemic immune response of Drosophila and are hereafter called DIMs for "Drosophila Immune-induced Molecules". DIMs are revealed by direct MALDI-TOF
MS analysis of the hemolymph of single flies, without any purification step. Their
precise function in this process remains to be established. For this, we first determined the most accurate molecular mass of the DIMs to see if some of these compounds correspond to already described molecules.
MALDI-TOP MS allows differential mass analysis of complex biological mixtures in order to compare the qualitative pattern of compounds present before and
after a physiological process.
5
Molecular Mass Characterization of the DIMs
At their ionization threshold, where the best resolution and mass accuracy are
obtained (Jensen et al. 1997), and with an external calibration, the masses of
peaks 9, 11, 17 and 19 correlate with the masses of already identified antimicrobial peptides which are secreted into the hemolymph after immune challenge. (i)
Peaks 9 and 11 (m/z measured at 2402.9 and 2566.1) correspond to the monocharged ions of the antibacterial O-glycosylated drosocin (Bulet et al. 1993) carrying an N-acetylgalactosamine (drosocin-lS, calculated molecular mass of
2401.9 Da) and an N-acetylgalactosamine-galactose (drosocin-2S, calculated
molecular mass of 2564.4 Da), respectively. (ii) Peak 17 at m/z 3046.6 corresponds
to one form of the antimicrobial metchnikowin with a calculated mass of 3045.4
Da (Levashina et al. 1995). (iii) Peak 19 at m/z 4890.6 corresponds to drosomycin
(calculated mass of 4889.5 Da), which exhibits potent antifungal activity (Fehlbaum et al. 1994).
However, not all the systemic antimicrobial peptides already characterized in
immune challenged Drosophila were detectable in the mass spectra recorded
from the hemolymph of immune-challenged flies (Fig. 11.2). Several hypotheses
can explain this discrepancy. For example, we did not observe the mass signal of
defensin, an antibacterial peptide of 4354 Da (Dimarcq et al. 1994), certainly due
to its very low concentration «2 [lM) in the hemolymph of immune-challenged
flies compared to the concentration of 100 [lM for drosomycin, 40 [lM for the two
glycoforms of drosocin and 10 [lM for metchnikowin. This hypothesis was verified since a clear mass signal was observed when purified Drosophila defensin
was added to the hemolymph of immune-challenged flies at a final concentration
of 10 [lM (data not shown). This result was surprising since MALDI-TOF MS is
considered to be highly sensitive (femtomole range). However, such a high sensitivity appears to be accessible only on purified or partially purified compounds.
In fact, we clearly observed that efficiency of MALDI-TOF MS, from the point of
view of sensitivity, decreases when a compound is analyzed in more and more
complex mixtures. This alteration in sensitivity can be the result of suppression
163
the 24 induced molecules do not result from bacteria. This result was confirmed
by the analysis of Drosophila mutants (see below).
In summary, none ofthe 24 induced molecules detected by MALDI-TOF MS in
Drosophila hemolymph are of bacterial origin or coming from the lysis of the
Drosophila blood cells. Therefore they represent molecules involved in the systemic immune response of Drosophila and are hereafter called DIMs for "Drosophila Immune-induced Molecules". DIMs are revealed by direct MALDI-TOF
MS analysis of the hemolymph of single flies, without any purification step. Their
precise function in this process remains to be established. For this, we first determined the most accurate molecular mass of the DIMs to see if some of these compounds correspond to already described molecules.
MALDI-TOP MS allows differential mass analysis of complex biological mixtures in order to compare the qualitative pattern of compounds present before and
after a physiological process.
5
Molecular Mass Characterization of the DIMs
At their ionization threshold, where the best resolution and mass accuracy are
obtained (Jensen et al. 1997), and with an external calibration, the masses of
peaks 9, 11, 17 and 19 correlate with the masses of already identified antimicrobial peptides which are secreted into the hemolymph after immune challenge. (i)
Peaks 9 and 11 (m/z measured at 2402.9 and 2566.1) correspond to the monocharged ions of the antibacterial O-glycosylated drosocin (Bulet et al. 1993) carrying an N-acetylgalactosamine (drosocin-lS, calculated molecular mass of
2401.9 Da) and an N-acetylgalactosamine-galactose (drosocin-2S, calculated
molecular mass of 2564.4 Da), respectively. (ii) Peak 17 at m/z 3046.6 corresponds
to one form of the antimicrobial metchnikowin with a calculated mass of 3045.4
Da (Levashina et al. 1995). (iii) Peak 19 at m/z 4890.6 corresponds to drosomycin
(calculated mass of 4889.5 Da), which exhibits potent antifungal activity (Fehlbaum et al. 1994).
However, not all the systemic antimicrobial peptides already characterized in
immune challenged Drosophila were detectable in the mass spectra recorded
from the hemolymph of immune-challenged flies (Fig. 11.2). Several hypotheses
can explain this discrepancy. For example, we did not observe the mass signal of
defensin, an antibacterial peptide of 4354 Da (Dimarcq et al. 1994), certainly due
to its very low concentration «2 [lM) in the hemolymph of immune-challenged
flies compared to the concentration of 100 [lM for drosomycin, 40 [lM for the two
glycoforms of drosocin and 10 [lM for metchnikowin. This hypothesis was verified since a clear mass signal was observed when purified Drosophila defensin
was added to the hemolymph of immune-challenged flies at a final concentration
of 10 [lM (data not shown). This result was surprising since MALDI-TOF MS is
considered to be highly sensitive (femtomole range). However, such a high sensitivity appears to be accessible only on purified or partially purified compounds.
In fact, we clearly observed that efficiency of MALDI-TOF MS, from the point of
view of sensitivity, decreases when a compound is analyzed in more and more
complex mixtures. This alteration in sensitivity can be the result of suppression