160
PH. BULET and S. UTTENWEILER-JOSEPH
3.2
Sample Preparation and Experimental Conditions of MALDI-TOF MS Analysis
of Drosophila hemolymph
Drosophila hemolymph was collected from a single fly using a glass capillary
(Fig. ll.lA) and directly deposited onto the MALDI target pretreated as
described below (Fig. ll.lB). There are many different sample preparations to
incorporate a protein mixture into matrix crystals such as the dried droplet, the
thin polycrystalline fIlm, the seeded microcrystalline fIlm or the thick layer
methods (for reviews, see Beavis and Chait 1996 and Kussmann et al. 1997). A
sample preparation including nitrocellulose (for further experimental details, see
Uttenweiler-Joseph et al. 1998) was preferred because nitrocellulose has the property to enhance the ionization yield of molecules as already described (Preston et
al. 1993, Liu et al. 1995, Uttenweiler-Joseph et al. 1997). In addition, this preparation allows an intensive washing of the final preparation before analysis resulting
in a better resolution and a higher mass accuracy due to the elimination of salts
adducts. Two different matrices were tested with the nitrocellulose preparation:
a-cyano-4-hydroxycinnamic acid (4-HCCA) usually used for the analysis of peptides, and sinapinic acid which is more selective for proteins. During this first
approach, all mass spectra were acquired in the positive linear mode with an
instrument equipped with a continuous extraction (no delayed-extraction). The
same pattern was obtained in the negative mode, but with a lower ionization efficiency (data not shown). Comparison of the mass spectra obtained with 4-HCCA
or with sinapinic acid as matrix reveals that sinapinic acid enhanced the signal
A
H
umpl. pre""nol;on
bed,,(
h
' "
c
~c.:, . . . iRllplnlC'add.u .. dUu.w IM-mol)mph
,
IUrlO"
,
I~I("I
mlz
Fig. 11.1. Experimental conditions for the analysis of hemolymph from an immune-challenged Drosophila. The hemolymph (0.1 f!l), collected from a single fly with a glass capillary (A), is directly analyzed by MALDI-TOF MS with a sample preparation using nitrocellulose (B) (for further details, see
Uttenweiler-Joseph et aI. 1998). In order to obtain the most qualitative mass spectrum, different
parameters were tested like the nature of the matrix [a-cyano-4-hydroxycinnamic acid (4-HCCA) or
sinapinic acid] and the matrixlanalyte ratio modified by hemolymph dilution (C)
PH. BULET and S. UTTENWEILER-JOSEPH
3.2
Sample Preparation and Experimental Conditions of MALDI-TOF MS Analysis
of Drosophila hemolymph
Drosophila hemolymph was collected from a single fly using a glass capillary
(Fig. ll.lA) and directly deposited onto the MALDI target pretreated as
described below (Fig. ll.lB). There are many different sample preparations to
incorporate a protein mixture into matrix crystals such as the dried droplet, the
thin polycrystalline fIlm, the seeded microcrystalline fIlm or the thick layer
methods (for reviews, see Beavis and Chait 1996 and Kussmann et al. 1997). A
sample preparation including nitrocellulose (for further experimental details, see
Uttenweiler-Joseph et al. 1998) was preferred because nitrocellulose has the property to enhance the ionization yield of molecules as already described (Preston et
al. 1993, Liu et al. 1995, Uttenweiler-Joseph et al. 1997). In addition, this preparation allows an intensive washing of the final preparation before analysis resulting
in a better resolution and a higher mass accuracy due to the elimination of salts
adducts. Two different matrices were tested with the nitrocellulose preparation:
a-cyano-4-hydroxycinnamic acid (4-HCCA) usually used for the analysis of peptides, and sinapinic acid which is more selective for proteins. During this first
approach, all mass spectra were acquired in the positive linear mode with an
instrument equipped with a continuous extraction (no delayed-extraction). The
same pattern was obtained in the negative mode, but with a lower ionization efficiency (data not shown). Comparison of the mass spectra obtained with 4-HCCA
or with sinapinic acid as matrix reveals that sinapinic acid enhanced the signal
A
H
umpl. pre""nol;on
bed,,(
h
' "
c
~c.:, . . . iRllplnlC'add.u .. dUu.w IM-mol)mph
,
IUrlO"
,
I~I("I
mlz
Fig. 11.1. Experimental conditions for the analysis of hemolymph from an immune-challenged Drosophila. The hemolymph (0.1 f!l), collected from a single fly with a glass capillary (A), is directly analyzed by MALDI-TOF MS with a sample preparation using nitrocellulose (B) (for further details, see
Uttenweiler-Joseph et aI. 1998). In order to obtain the most qualitative mass spectrum, different
parameters were tested like the nature of the matrix [a-cyano-4-hydroxycinnamic acid (4-HCCA) or
sinapinic acid] and the matrixlanalyte ratio modified by hemolymph dilution (C)