A MALDI-TOF Mass Spectrometry Approach to Investigate the Defense Reactions
161
intensity of the compounds with a molecular mass higher than 9 kDa (Fig_
1 LIC)_ Nevertheless, with sinapinic acid, some compounds with a low molecular
mass were undetectable, and resolution as well as mass accuracy were decreasing
compared to the results observed with 4-HCCA_ To be as qualitative as possible,
the matrix 4-HCCA was preferred to sinapinic acid_
For MALDI MS, the matrixlanalyte ratio is important as it influences the formation of the co-crystals on which depends the quality of the mass spectra. To
analyze this parameter, different dilutions were performed on Drosophila hemolymph (10 and 100-fold in acidic water). Compared to crude hemolymph, a 10fold dilution had the effect of dramatically decreasing the signal to noise ratio
resulting in a loss of resolution and mass accuracy (Fig. 11.1 C). Furthermore, no
additional peak was visible. Consequently, our subsequent investigations were
performed directly on undiluted Drosophila hemolymph with a sample preparation including nitrocellulose and 4-HCCA as a matrix. Finally, only the mass
range from L5 to 11 kDa was investigated because the mass signal intensity was
very low above this mass range resulting in a lack of reproducibility.
We would also like to stress the importance of the laser power intensity. In
fact, we clearly observed from these preliminary experiments, that quality of
mass spectra is dependent on laser power intensity. A higher laser power intensity increased the number of peaks detectable. However, when laser power intensity is higher than the ionization threshold, resolution and mass accuracy
decrease. For the purpose of this study, laser power intensity was first adjusted to
a level allowing the detection of the highest number of compounds as illustrated
in figure 1 L 1 C and secondly to a level corresponding to the ionization threshold
of any individual compound detected for the determination of their most accurate mass.
The best conditions of MALDI- TOP MS analysis must be found for each complex
biological mixture with regard to the aims (qualitative analysis versus detection of
a specific compound) and the requirements for reproducibility, mass resolution
and mass accuracy.
4
Differential Analysis of Hemolymph from Immune-Challenged
and Unchallenged Drosophila by MALDI-TOF MS
The mass spectrum obtained from the hemolymph of a 24h-immune-challenged
fly was compared to that of a control (unchallenged) fly (Fig. 11.2). In the conditions mentioned above and whatever the sex of the fly, the mass spectra were
highly reproducible for m/z values between 1,500 and 11,000 (more than 50 control and immune-challenged flies were individually analyzed).
Mass spectrum of the hemolymph of immune-challenged Drosophila is more
complex than that of controls. More precisely, in addition to compounds already
found in control flies, 24 additional molecules (numbered from 1 to 24 in Fig.
lL2) were clearly present in immune-challenged hemolymph.
The 24 molecules detected after injury could be systemic molecules or cytoplasmic components released by the Drosophila blood cells which can be lysed by
the acidity of the MALDI matrix solution used. To distinguish between these two
161
intensity of the compounds with a molecular mass higher than 9 kDa (Fig_
1 LIC)_ Nevertheless, with sinapinic acid, some compounds with a low molecular
mass were undetectable, and resolution as well as mass accuracy were decreasing
compared to the results observed with 4-HCCA_ To be as qualitative as possible,
the matrix 4-HCCA was preferred to sinapinic acid_
For MALDI MS, the matrixlanalyte ratio is important as it influences the formation of the co-crystals on which depends the quality of the mass spectra. To
analyze this parameter, different dilutions were performed on Drosophila hemolymph (10 and 100-fold in acidic water). Compared to crude hemolymph, a 10fold dilution had the effect of dramatically decreasing the signal to noise ratio
resulting in a loss of resolution and mass accuracy (Fig. 11.1 C). Furthermore, no
additional peak was visible. Consequently, our subsequent investigations were
performed directly on undiluted Drosophila hemolymph with a sample preparation including nitrocellulose and 4-HCCA as a matrix. Finally, only the mass
range from L5 to 11 kDa was investigated because the mass signal intensity was
very low above this mass range resulting in a lack of reproducibility.
We would also like to stress the importance of the laser power intensity. In
fact, we clearly observed from these preliminary experiments, that quality of
mass spectra is dependent on laser power intensity. A higher laser power intensity increased the number of peaks detectable. However, when laser power intensity is higher than the ionization threshold, resolution and mass accuracy
decrease. For the purpose of this study, laser power intensity was first adjusted to
a level allowing the detection of the highest number of compounds as illustrated
in figure 1 L 1 C and secondly to a level corresponding to the ionization threshold
of any individual compound detected for the determination of their most accurate mass.
The best conditions of MALDI- TOP MS analysis must be found for each complex
biological mixture with regard to the aims (qualitative analysis versus detection of
a specific compound) and the requirements for reproducibility, mass resolution
and mass accuracy.
4
Differential Analysis of Hemolymph from Immune-Challenged
and Unchallenged Drosophila by MALDI-TOF MS
The mass spectrum obtained from the hemolymph of a 24h-immune-challenged
fly was compared to that of a control (unchallenged) fly (Fig. 11.2). In the conditions mentioned above and whatever the sex of the fly, the mass spectra were
highly reproducible for m/z values between 1,500 and 11,000 (more than 50 control and immune-challenged flies were individually analyzed).
Mass spectrum of the hemolymph of immune-challenged Drosophila is more
complex than that of controls. More precisely, in addition to compounds already
found in control flies, 24 additional molecules (numbered from 1 to 24 in Fig.
lL2) were clearly present in immune-challenged hemolymph.
The 24 molecules detected after injury could be systemic molecules or cytoplasmic components released by the Drosophila blood cells which can be lysed by
the acidity of the MALDI matrix solution used. To distinguish between these two