A MALDI-TOF Mass Spectrometry Approach to Investigate the Defense Reactions
159
During the last years, matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) has appeared as a powerful technique to analyze complex mixtures of peptide,S within different biological samples. The performance of MALDI -TOF MS is explained by its sensitivity (femtomolar to attomolar concentrations), its tolerance towards buffers and salts, and
by the ionization process that chiefly produces single-charged ions leading to
clear spectra (for review, see Burlingame et al. 1998). Consequently, MALDI-TOF
MS is the most suitable MS technique for the direct analysis of complex biological
samples such as cell lines (van Adrichem et al. 1998), thin-layer preparations of
tissues (Caprioli et al. 1997), single giant neurons (Jimenez et al. 1994, Li et al.
1994), procaryotic organisms (Claydon et al. 1996, Easterling et al. 1998), or eucaryotic cells (Stahl et al. 1997, Redeker et al. 1998). In these studies, MALDI-TOF
MS allowed the establishment of peptide profiles which served to obtain characteristic fingerprints or to follow the synthesis or the maturation processing of
bioactive peptides.
In the present contribution, MALDI -TOF MS differential display of the hemolymph of individual immune-challenged versus unchallenged Drosophila led to
the detection and molecular mass characterization of new molecules involved in
insect immunity. This strategy, as developed for Drosophila, can be applied to
other biological systems for the direct identification from complex biological
samples of compounds induced or repressed during a given physiological process, regardless of their activities.
3
Measuring Molecular Masses in Complex Biological Samples:
Establishment of Experimental Conditions
3.1
Critical Points for MALDI-TOF MS Analysis of Complex Mixtures
Briefly, for MALDI analysis, the analyte is mixed with an excess (10 3 to lOs-fold)
of "matrix" to form co-crystals. The matrix serves to isolate the analyte molecules from each other and to absorb the intense laser radiation used to vaporize
and propel the analyte molecules into the gas phase where they are ionized.
Matrix is clearly the most important factor to obtain a mass signal, but many
other parameters influence the quality of the mass spectrum, like the sample
preparation (how the co-crystals are formed), the matrix to analyte ratio and the
laser power (Cohen and Chait 1996, Kussmann et al. 1997, Roepstorff et al. 1998).
In a previous study, we observed that all these parameters are critical when
working on complex mixtures of peptides and proteins (Uttenweiler-Joseph et al.
1997). With regard to this observation, our first step in establishing the protocol
for MALDI-TOF MS analysis of Drosophila hemolymph was to determine the
optimal conditions to obtain high quality spectra.
159
During the last years, matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) has appeared as a powerful technique to analyze complex mixtures of peptide,S within different biological samples. The performance of MALDI -TOF MS is explained by its sensitivity (femtomolar to attomolar concentrations), its tolerance towards buffers and salts, and
by the ionization process that chiefly produces single-charged ions leading to
clear spectra (for review, see Burlingame et al. 1998). Consequently, MALDI-TOF
MS is the most suitable MS technique for the direct analysis of complex biological
samples such as cell lines (van Adrichem et al. 1998), thin-layer preparations of
tissues (Caprioli et al. 1997), single giant neurons (Jimenez et al. 1994, Li et al.
1994), procaryotic organisms (Claydon et al. 1996, Easterling et al. 1998), or eucaryotic cells (Stahl et al. 1997, Redeker et al. 1998). In these studies, MALDI-TOF
MS allowed the establishment of peptide profiles which served to obtain characteristic fingerprints or to follow the synthesis or the maturation processing of
bioactive peptides.
In the present contribution, MALDI -TOF MS differential display of the hemolymph of individual immune-challenged versus unchallenged Drosophila led to
the detection and molecular mass characterization of new molecules involved in
insect immunity. This strategy, as developed for Drosophila, can be applied to
other biological systems for the direct identification from complex biological
samples of compounds induced or repressed during a given physiological process, regardless of their activities.
3
Measuring Molecular Masses in Complex Biological Samples:
Establishment of Experimental Conditions
3.1
Critical Points for MALDI-TOF MS Analysis of Complex Mixtures
Briefly, for MALDI analysis, the analyte is mixed with an excess (10 3 to lOs-fold)
of "matrix" to form co-crystals. The matrix serves to isolate the analyte molecules from each other and to absorb the intense laser radiation used to vaporize
and propel the analyte molecules into the gas phase where they are ionized.
Matrix is clearly the most important factor to obtain a mass signal, but many
other parameters influence the quality of the mass spectrum, like the sample
preparation (how the co-crystals are formed), the matrix to analyte ratio and the
laser power (Cohen and Chait 1996, Kussmann et al. 1997, Roepstorff et al. 1998).
In a previous study, we observed that all these parameters are critical when
working on complex mixtures of peptides and proteins (Uttenweiler-Joseph et al.
1997). With regard to this observation, our first step in establishing the protocol
for MALDI-TOF MS analysis of Drosophila hemolymph was to determine the
optimal conditions to obtain high quality spectra.