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De Novo Sequencing of an Immune-Induced Peptide of Drosophila Melanogaster
N. Carte et al.,Eur.J. Mass Spectrom.7, 399–408 (2001)
De novo sequencing by nano-electrospray
multiple-stage tandem mass spectrometry
of an immune-induced peptide of
Drosophila melanogaster
Nathalie Carte, Nukhet Cavusoglu, Emmanuelle Leize*and Alain Van Dorsselaer
Laboratoire de Spectrométrie de Masse Bio-Organique, UMR 7509, ECPM, 25 rue Becquerel, 67087 Strasbourg Cedex, France.
E-mail: leize@chimie.u-strasbg.fr
Maurice Charlet and Philippe Bulet*
Institut de Biologie Moléculaire et Cellulaire, UPR 9022, CNRS, “Réponse Immunitaire et Développement chez les Insectes”,
15 rue René Descartes, 67084 Strasbourg Cedex, France. E-mail: P.Bulet@ibmc.u-strasbg.fr
To combat infection, the fruit fly Drosophila melanogaster responds by rapid synthesis of a series of immune-induced molecules
reported as Drosophila immune-induced molecules (DIMs). Characterization of the primary structure of the DIMs is required to
establish their exact function. In order to get such information, previous studies on the elucidation of primary structures of the DIMs
were developed using a methodology combining matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), high-
performance liquid chromatography (HPLC), enzymatic digestion and Edman degradation. Nevertheless, some of the DIMs were
resistant to classical Edman sequencing. Therefore, mass spectrometry was used to characterize the primary structure of one of the
DIMs, namely the N-blocked DIM13 peptide. The complete sequence of DIM13 was established by means of a strategy of nano-
electrospray ionisation (ESI) combined with multiple-stage tandem mass spectrometry (MSn) and then was partially confirmed with
a combination of enzymatic digestions and MALDI-MS analyses. Interestingly, most of the amino acid sequences have been deter-
mined using three-stage (MS3) and four-stage (MS4) tandem experiments, whereas classical tandem mass spectrometry (MS2) yielded
only incomplete sequence information. Finally, DIM13 is a 23 amino acid peptide with a pyroglutamic modification at the N terminal
position. This work illustrates the remarkable advantages of MS3and MS4compared with the MS2experiment for de novo peptide
sequencing. The use of nano-ESI also makes these experiments compatible with the low amount (picomolar level) of DIM13 peptide
available for sequencing by ESI-MSn.
Keywords: nano-electrospray, ion trap MSn,de novo sequencing, Drosophila melanogaster, innate immunity
Introduction
The fruit fly Drosophila melanogaster, like other
insects, is particularly resistant to microbial infections.
Three main innate immune mechanisms are considered to
contribute to this resistance: (1) cellular reactions allowing
phagocytosis and encapsulation of the microorganisms by
the insect blood cells (hemocytes), (2) activation of
proteolytic cascades leading to melanization, coagulation
and opsonization, and (3) transient activation of the synthe-
sis of antimicrobial peptides.1Recent advances in the molec-
ular mechanisms of innate host defence reactions in
multicellular organisms from mammals to insects, including
Drosophila, have outlined important similarities. For this
reason and because of the potency of Drosophila for genetic
studies, this model appears as a powerful tool to study the
evolution of the mechanisms of innate immunity. Although
the structure and the regulated expression of the
antimicrobial peptides have been intensively studied during
the past ten years,2,3 only fragmentary data are available on
the other immune processes developed by the fruit fly to
fight off an infection. In order to find new Drosophila
immune molecules, not related to antimicrobial substances,
a protocol consisting of a differential display by matrix-
© IM Publications 2001, ISSN 1356-1049
N. Carte et al.,Eur.J. Mass Spectrom.7, 399–408 (2001) 399
assisted laser desorption/ionization mass spectrometry
(MALDI-MS) and high-performance liquid chromatogra-
phy (HPLC) has been optimized on Drosophila hemolymph
(insect blood) collected from experimentally infected and
non-infected flies.4MALDI-MS analysis led to the detection
of at least 24 immune-induced molecules (DIMs, i.e.
Drosophila immune-induced molecules) in the Drosophila
hemolymph, 24 hours following an experimental infection.4,5
Some of the DIMs were resistant to classical Edman
sequencing degradation, for example DIM13, which was
found to be N-terminal blocked. Moreover, the chromato-
graphic fraction of DIM13 was not pure enough for use of
the classical approach, which comprises enzymatic cleavage
of the peptide followed by tandem mass spectrometry
(MS/MS) experiments of the resulting lower mass peptides.
A better purity could be achieved, but to the detriment of the
amount of DIM13 available for the sequence determination.
In our study, we intended to demonstrate that a strategy rely-
ing on the multiple-stage tandem mass spectrometry (MSn)
capabilities of an ion trap mass spectrometer6could be used
to determine de novo sequence of the entire DIM13 peptide.
For the structural elucidation of the DIM13 peptide, three-
stage (MS3) and four-stage (MS4) experiments performed
with an ion trap mass spectrometer yielded significant and
relevant sequence information to fully characterize this N-
blocked peptide.
As we were sequencing a novel peptide, further analy-
ses, such as reduction, alkylation and enzymatic digestion
using a carboxypeptidase followed by MALDI-MS were
performed to partially validate the sequence of DIM13 pep-
tide determined by electrospray ionzisation (ESI)-MSn.In
order to perform all the ESI-MSnexperiments with the esti-
mated 10 pmoles of DIM13 available, we took advantage of
the low consumption of analyte using the coupling of the
nano-electrospray source with an ion trap mass analyzer.7–9
The elucidation of the primary structure of this 23 amino
acid peptide DIM13 is presented here in detail.
Material and methods
Insect immunization and hemolymph collection
The insect immunization and blood collection were per-
formed according to the procedure previously described.4
HPLC separation and Edman degradation
The acidified hemolymph collected from 180 flies was
subjected to separation using reversed-phase (RP)-HPLC on
an Aquapore RP 300 C8column (1 × 100 mm). Separation
was performed with a linear gradient of 0–80% acetonitrile
in acidified water [0.05% trifluoroaceticacid (TFA)] over
80 min at a flow rate of 80 µL min–1 at 35°C. The fractions,
which were collected by hand in low-protein-binding
Eppendorf tubes, were analyzed by MALDI-MS in order to
detect the fraction containing DIM13. A second purification
step was performed with a linear gradient from 2% to 22%
acetonitrile in acidified water over 10 min and from 22% to
37% in 75 min at a flow rate of 80 µL min–1 on the same col-
umn as previously at 35°C. The HPLC fraction, which did
not contain the DIM13 peptide exclusively, was estimated,
according to its optical density, at 40 pmoles. This estima-
tion was done by comparison with the optical density of pep-
tides of the same family. We divided the total amount in
three aliquots: one for the Edman degradation (10 pmoles),
one for the nano-ES-MSnexperiments (10 pmoles) and the
last one for enzymatic digestions and MALDI-MS analyses
(20 pmoles).
An Edman degradation experiment was attempted on
the HPLC fraction containing mainly DIM13. Automated
Edman degradation of DIM13 was performed on a pulse liq-
uid automatic sequenator (Applied Biosystems ABI473A).
Nano-electrospray multiple-stage tandem mass spectrometry
(NanoES-MSn)
Sample preparation
To avoid the inconvenience of the TFA present in the
HPLC sample during mass spectrometric analyses, the dis-
solved DIM13 (10 pmoles estimated) was loaded on a C18
Zip Tip (Millipore) and concentrated in 2 µL H2O+CH
3CN
(50 : 50 with 1% formic acid). All tandem mass spectromet-
ric analyses were performed with a total volume of 2 µL of a
concentrated DIM13 solution, deposited on a previously
rinsed nanospray capillary.
Electrospray instrumentation
Low-energy collisions of multiply-charged ions were
performed on an ion trap mass spectrometer (ESQUIRE-LC,
Bruker–Franzen Analytik GmbH, Germany) equipped with
a nanospray source. The gold/palladium-coated nanospray
capillaries were from Protana (Odense, Denmark). The cap-
illary and the counter-electrode voltages were set to 750 V
and 250 V, respectively. The voltage applied on the exit of
the metallized-glass capillary interface was optimized at
80 V. The voltage applied on skimmer 1 was optimized at
40 V.
Calibration of the mass analyzer was performed with
the multiply-charged ions of the following five standard
peptides: leu-enkephalin, angiotensin, substance P,
bombesin, and ACTH, having monoisotopic molecular
weights of 711.38, 1045.54, 1346.74, 1619.81 and
2464.20 Da, respectively.
To perform tandem mass spectrometry experiments
(MSn), isolation of the precursor ion was achieved by scan-
ning frequencies of ions to eject all other ions from the trap.
The precursor ion was fragmented by applying a resonance
frequency on the end cap electrodes (peak-to-peak ampli-
tude of 0.8 to 2.5 V) matching the frequency of the selected
ion. As a result, the kinetic energy of the precursor ion
increases and dissociation, due to collisions with the helium
buffer gas (pressure of 5 × 10–3 mbar) occurs. Sequences of
isolation and fragmentation were repeated for MSnexperi-
ments to gain structural information on the selected frag-
400 De Novo Sequencing of an Immune-Induced Peptide of Drosophila Melanogaster
ment ions. In this study, to obtain MSn+1 (n=1,2,3)
fragmentation mass spectra of a good quality, the precursor
ion was chosen according to its intensity on the different MSn
or MSn–1 spectra. Ions were scanned in standard resolution
mode with a scan speed of 13,000 m/z per second. A total of
20 scans were averaged to obtain a mass spectrum.
Matrix-assisted laser desorption/ionization mass spectrometry
(MALDI-MS)
Sample reduction and alkylation
About 20 pmoles of DIM13 was dissolved in 25 mM
NH4HCO3buffer at pH 8. First, the peptide was reduced in
the presence of 10 mM of Dithiothreitol (DTT, Sigma, St
Louis, USA) in a 200-molar excess at 57°C for one hour.
Second, alkylation was performed with a 240-molar excess
of 55 mM iodoacetamide (Sigma, St Louis, USA) as
alkylating agent.
Trypsin digestion
An aliquot of the reduced and alkylated DIM13
(10 pmoles) dissolved in 25 mM NH4HCO3buffer at pH 8
was subjected to trypsin digestion (sequencing-grade
trypsin, Promega) using an enzyme-to-substrate ratio of
1 : 20 (w / w). 0.5 µL of this solution was deposited on the
MALDI probe after two hours for overnight incubation at
35°C.
Carboxypeptidase P digestion
An aliquot of the reduced and alkylated DIM13
(10 pmoles) dissolved in 25 mM ammonium carbonate
buffer at pH 8, was treated with carboxypeptidase P
(Boehringer, Mannheim, Germany; initially stored in
25 mM sodium citrate) at an enzyme-to-substrate ratio of
1:20(w/w).
10–12 0.5 µL of the product was deposited on a
MALDI probe (see following section) after 30 s, 2 min,
5 min, 10 min and 48 h of incubation at 37°C. This C-termi-
nal sequencing by carboxypeptidase P was followed by
MALDI-MS and gave optimal sequencing data after 48
hours of incubation.
Sample deposition for MALDI analysis
The thin-layer sample preparation method was used for
MALDI-MS analysis. Briefly, 0.5 µL of α-cyano-4-
hydroxycinnamic acid (HCCA, Sigma; 7 g L–1 in acetone)
was placed on the probe tip. When this was dry, 0.5 µL of 1%
TFA was deposited on the crystallized matrix bed, followed
by the deposition of 0.5 µL of digestion mixture loaded at
different time intervals. The acidity of the matrix and of the
1% TFA droplet was sufficient to completely quench any
further digestion. After drying, the target was washed with
2 µL of 1% aqueous TFA, the liquid removed after a few sec-
onds using forced air and the sample again dried under vac-
uum.
MALDI instrumentation
This study was carried out on a BIFLEX matrix-assisted
laser desorption/ionization time-of-flight mass spectrometer
(Bruker, Bremen, Germany), equipped with SCOUT high-
resolution optics and a gridless reflectron. This instrument
has a maximum acceleration potential of 20 kV and may be
operated in either linear or reflectron mode. Ionization is
accomplished with the 337 nm beam from a nitrogen laser
with a repetition rate of 3 Hz. A camera mounted on a micro-
scope allows visualization of the sample crystallization
homogeneity before measurements. The spectra were
acquired in the positive-ionization mode using both the lin-
ear and reflectron modes. External calibration was per-
formed with five standard peptides: leu-enkephalin,
angiotensin, substance P, bombesin and ACTH, with aver-
age/monoisotopic molecular weights of 711.82 / 711.38,
1046.2 / 1045.54, 1347.66 / 1346.74, 1620.86 / 1619.81 and
2465.71 / 2464.20 Da, respectively.
Results and discussion
The strategy used to determine the de novo sequence of
the entire Drosophila melanogaster immune-induced pep-
tide DIM13, relied on the MSncapabilities of the ion trap
mass spectrometer. Thus, MALDI-MS was used on peptide
digests to confirm the determined sequence. Additional
information on DIM13 was also obtained after reduc-
tion–alkylation experiments, indicating that no cysteine resi-
due was present in the peptide sequence. The nano-ES
spectrum of the HPLC fraction containing DIM13 displayed
three major charge states at m/z 1325.7 (2+) (monoisotopic
mass), m/z 884.3 (3+) and m/z 663.8 (4+) (average values)
giving a measured monoisotopic weight value for DIM13 of
2649.4 ± 0.2 Da (Figure 1).
In agreement with the mobile-proton model,13 different
collision-induced dissociation (CID) processes using ES-
MSnare known to occur according to the charge state of the
N. Carte et al.,Eur.J. Mass Spectrom.7, 399–408 (2001) 401
Figure 1. NanoES-MS spectrum of the DIM13 peptide dissolved
in H2O+CH
3CN (50 : 50) with 1% of HCOOH.
peptide precursor ion.14 Consequently, MS2spectra were per-
formed successively on each multiply-charged ion observed
in the DIM13 nano-ES spectrum to obtain as much structural
information as possible.
The MS2spectrum of the (2+) molecular ion (m/z
1325.7) [Figure 2(a)] displayed 10 significant singly- (1+)
and doubly- (2+) charged fragment ions (Table 1). The C-ter-
minal amino acid, a phenylalanine, was recognised by inter-
pretation of the (2+) fragment ions at m/z 1243.1, m/z 1234.2
and m/z 1225.1, corresponding respectively to b22,b
22–H2O
and b22–2H2O, and was further confirmed by the fragmenta-
tion of the (3+) molecular ion (m/z 884.3). The phenylalanine
was placed at the C-terminus since the N-terminus of the
DIM13 is expected to be blocked on the basis of the failed
attempt at Edman degradation.
The MS2spectrum of the (3+) molecular ion (m/z 884.3)
[Figure 2(b)] gave 11 significant fragments ions (Table 1)
whose interpretation did not allow any further sequence
402 De Novo Sequencing of an Immune-Induced Peptide of Drosophila Melanogaster
Figure 2. MS2fragment-ion spectra obtained from the DIM13 peptide : (a) (2+) molecular ion at m/z 1325.7, (b) (3+) molecular ion at
m/z 884.3 and (c) (4+) molecular ion at m/z 663.8.
N. Carte et al.,Eur.J. Mass Spectrom.7, 399–408 (2001) 403
Fragments by MS2Fragments by MS3Fragments by MS4
Parent ion: 1325.7 (2+)
1764.7 (1+) b15–NH3
1658.8 (1+) y15
1463.7 (1+) b12
1307.7 (2+) M+2H+–2H2O
1243.1 (2+) b22
1234.2 (2+) b22H2O
1225.1 (2+) b22–2H2O
1187.6 (1+) y11
992.3 (1+) b8
869.5 (1+) y8
Parent ion: 992.3 (1+) b8
974.3 (1+) b8–H2O
920.3 (1+) a8–CO2
894.4 (1+) c7
877.2 (1+) b7
780.3 (1+) b7
763.3 (1+) b6–NH3
734.4 (1+) HVERPD
624.4 (1+) b5
597.3 (1+) VERPD
580.3 (1+) VERPD–H2O
495.2 (1+) b4
467.2 (1+) a4
396.2 (1+) b3
368.2 (1+) a4
Parent ion: 1187.6 (1+) y11
1170.6 (1+)y11–NH3
1040.5 (1+) y10
1022.4 (1+) y10–H2O
983.5 (1+) y9
966.4 (1+) y9–NH3
869.4 (1+) y8
812.3 (1+) y7
720.2 (1+) FGNGGFSA–H2O
Parent ion: 396.2 (1+) b3
379.9 (1+) b3–17
368.0 (1+) a3
352.2 (1+) a3–16
241.7 (1+) b2–H2O
(ϕ–CH=CH–NH–CH2–CH2–
imidazole+H+)
Parent ion: 884.3 (3+)
1325.6 (2+) M+2H+
1187.6 (1+) y11
1040.4 (1+) y10
992.3 (1+) b8
948.3 (2+) b17
846.7 (3+) M+3H+–Z
834.3 (2+) b14
829.7 (2+) y15
732.3 (2+) b12
624.2 (1+) b5
472.2 (1+) RTVD
Parent ion: 732.3 (2+) b12
992.3 (1+) b8
840.3 (1+) RPDRTVD
822.2 (1+) RPDRTVD–H2O
780.3 (1+) b6
723.2 (2+) b12–H2O
683.3 (2+) c11
674.8 (2+) b11
660.8 (2+) a11
624.2 (1+) b5
616.2 (2+) b10–H2O
603.2 (2+) HVERPDRTVD
495.2 (1+) b4
472.2 (1+) RTVD
467.2 (1+) a4
396.1 (1+) b3
Parent ion: 472.2 (1+) RTVD
428.2 (1+) RTVD–CO2
400.3 RTVD–CO2–CO
357.2(1+) RTV
258.2 (1+)RT
Parent ion: 846.7 (3+)
1237.5 (1+)b
*
11 (b11–Z)
1187.6 (1+)y
11
1119.4 (2+)y
20–NH3
1019.7 (1+)b
*
9–H2O(b9–H2O–Z)
973.2 (1+) HVERPDRT–H2O
934.7 (1+) VDFGNGGFSA–NH3
DRTVDFGNG–CO
679 (2+)b
*
12 (b12–Z)
Parent ion: 663.8 (4+)
1021.9 (2+) b18
948.3 (2+) b17
919.8 (2+) b16
891.3 (2+) b15
884.5 (3+) M+3H+
842.7 (2+) c14
834.2 (2+) b14
829.4 (3+) b22
777.1 (3+) b21
755.4 (1+) y6
752.2 (3+) y20
734.7 (3+) b20
711.2 (3+) b19
682.1 (3+) b18
659.4 (4+) M + H+–H2O
632.8 (3+) b17
613.8 (3+) b16
608.3 (1+) y5
594.8 (3+) b15
521.3 (1+) y4
450.3 (1+) y3
Parent ion: 948.3 (2+) b17
1401.7 (1+) ERPDRTVDFGNGG
1383.4 (1+) ERPDRTVDFGNGG–H2O
1272.6 (1+) RPDRTVDFGNGG
1254.6 (1+) VERPDRTVDFG–H2O
1197.6 (1+) VERPDRTVDF–H2O
1116.3 (1+) PDRTVDFGNGG
1099.6 (1+) PDRTVDFGNGG–NH3
1098.4 (1+) PDRTVDFGNGG–H2O
1019.4 (1+) DRTVDFGNGG
1002.4 (1+) PDRTVDFGN
992.3 (1+) b8
974.3 (1+) b8–H2O
PDRTVDFGN–28
934.7 (1+) DRTVDFGNG –28
904.3 (1+) RTVDFGNGG
894.4 (1+) c7
887.3 (1+) RTVDFGNGG–NH3
DRTVDFGN–H2O
780.3 (1+) b6
732.3 (2+) b12
723.3 (2+) b12–H2O
713.8 (2+)
VERPDRTVDFGNGG–NH3
701.3 (2+) ERPDRTVDFGNGG
683.3 (2+) c11
674.8 (2+) b11
665.8 (2+) b11–H2O
624.2 (1+) b5
495.2 (1+) b4
467.2 (1+) a4
396.1 (1+) b3
Table 1. Masses and interpretations of DIM13 fragments obtained with MS2,MS
3and MS4experiments.
determination. Nevertheless, a triply-charged fragment ion
at m/z 846.7, corresponding to the loss of 112.8 Da from the
molecular mass of the DIM13 peptide, was submitted to col-
lision-induced dissociation since the results revealed a
pyroglutamic acid located on the N-terminal end of the
DIM13 peptide (vide infra for MS3on the m/z 846.7 frag-
ment confirming the N-termination).
Finally, the MS2spectrum obtained for the fragmenta-
tion of the 4+molecular ion (m/z 663.8) was more complex
[Figure 2(c)] and displayed singly-, doubly- and triply-
charged fragment ions (Table 1). Sets of ions displaying the
same charge state were, however, able to provide internal
sequence information. Thus, the F18 S19 A20 tag was identified
by interpretation of the (1+) fragment ions. From the (2+)
fragment ions, the internal sequence N15 G16 G17 F18 could be
deduced. Finally, the (3+) fragment-ion series allowed eluci-
dation of the sequence at the C-terminal end. The C-terminal
sequence of DIM13 was found to be in agreement with the
former internal sequence tags determined from MS2spectra.
According to the whole MS2results, the C-terminal
sequence of the DIM13 was identified as (b14= 1667.4) – N15
G16 G17 F18 S19 A20 Q/K21 R22 F23 [Scheme 1(a)].
The DIM13 sequence orientation (N- or C-terminus)
deduced by MS2was confirmed by carboxypeptidase P treat-
ment and analysis by MALDI-MS. To follow the C-terminus
enzymatic sequencing, the kinetics of digestion were estab-
lished (see Experimental). Figure 3 shows the MALDI mass
spectrum from the sequential degradation of DIM13.
Carboxypeptidase treatment on the singly-charged (1+)
molecular ion of DIM13 at m/z 2650.0 (average value) gave
a first peak at m/z 2502.9 corresponding to a loss of 147.1 Da
confirming the presence of a phenylalanine as the C-termi-
nal amino acid. The successive mass losses of 156.0, 127.9,
70.8 and 87.0 Da confirmed the C-terminal sequence for
DIM13 as S19 A20 Q/K21 R22 F23.
To identify the complete sequence of DIM13, the deter-
mined C-terminal sequence (b14=1667.4)–N15 G16 G17 F18 S19
A20 Q/K21 R22 F23 was searched in protein databases
(Proteinprospector http://prospector.ucsf.edu). No success-
ful identification, at the date of the search (September 1999),
404 De Novo Sequencing of an Immune-Induced Peptide of Drosophila Melanogaster
Scheme 1. DIM13 sequence established (a) from MS2spectrum of the (4+) molecular ion at m/z 663.8 and (b) from MS2spectrum of
the (3+) molecular ion at m/z 884.3. Note that bold annotations represent the main cleavages.
Figure 3. MALDI-MS mass spectrum of the carboxypeptidase
digestion of DIM13 after 48 hours of incubation. Note that the
observed sodium adducts were due to the sodium citrate
buffer in which the carboxypeptidase P was initially stored.
was found. To characterize the full primary sequence of this
novel peptide, MS3and MS4tandem mass spectrometry was
required to generate sequence information from the
uncharacterized (N-terminal) region of the DIM13 peptide.
The MS3experiments performed on the (1+) precursor
ion at m/z 992.3 essentially generated N-terminal fragment
ions [Figure 4(a) and Table1] from which a partial sequence
on the N-terminal side of DIM13 was obtained, viz.
(b3=396.2)–V4E5R6P7D8[Scheme 2(a)]. The loss of CO2
from the a8ion corroborates the presence of acidic amino
acids (D and/or E) in the fragment at m/z 992.3. In the same
manner, ion b6loses NH3and, therefore, indicates a basic
amino acid such as the arginine located in the fragment at
m/z 992.3. Moreover, low intensity (1+) ions at m/z 734.4,
m/z 597.3, m/z 580.3 were identified as internal fragments of
the precursor at m/z 992.3 and indicated the presence of a
histidine residue on the N-terminal side of DIM13 (H3V4E5
R6P7D8).
N. Carte et al.,Eur.J. Mass Spectrom.7, 399–408 (2001) 405
Figure 4. MS3fragment ion spectra obtained from the DIM13 peptide : (a) (1+) precursor ion at m/z 992.3, (b) (2+) precursor ion at
m/z 948.3 and (c) (1+) precursor ion at m/z 1187.6.
In addition, MS3analysis performed on the (2+) ion at
m/z 948.3 [Figure 4(b) and Table 1] led to three major frag-
ments at m/z 992.3 (1+), m/z 904.3 (1+) and m/z 732.3 (2+). It
is important to notice for the further attribution of minor ions
that the fragmentation of the (2+) precursor ion at m/z 948.3
yields the two (1+) fragment ions at m/z 992.3 and m/z 904.3
by the same peptide bond cleavage [Scheme 2(b)]. Thus,
minor ions, previously identified as the daughter ions of m/z
992.3, attend their complementary fragment ions from the
m/z 948.3 doubly-charged precursor ion [Scheme 2(b)]. This
attribution unequivocally confirmed the sequence on the N-
terminal side.
MS3analysis performed on the (2+) precursor ion at m/z
732.3 exhibited one main cleavage giving two complemen-
tary (1+) fragment ions at m/z 992.3 and m/z 472.2. In
addition, the series of b ions, formerly characterized as
daughter ions of m/z 992.3 [Scheme 3(a)], was also
observed. Minor fragments were further interpreted thanks
to the elucidation of the previously uncharacterized (1+)
fragment ion at m/z 472.2.
For sensitivity reasons, MS4could not be performed on
this fragment ion at m/z 472.2. So, the precursor ion at m/z
472.2 was directly isolated from the MS2of the (3+) molecu-
lar ion at m/z 884.3, which also displayed a (2+) fragment ion
at m/z 732.3 and a (1+) fragment ion m/z 992.3. Therefore, the
isolated ion at m/z 472.2 was presumed to be obtained by a
double fragmentation resulting from the successive cleav-
ages of m/z 884.3 and m/z 732.3. The MS3experiment per-
406 De Novo Sequencing of an Immune-Induced Peptide of Drosophila Melanogaster
Scheme 3. DIM13 sequence established from MS3spectra of precursors: (a) (2+) fragment ion at m/z 732.3, (b) (1+) fragment ion at
m/z 472.2 and (c) (1+) fragment ion at m/z 1187.6.
Scheme 2. DIM13 sequence established from MS3spectra of precursors: (a) (1+) fragment ion at m/z 992.3, (b) (2+) fragment ion at
m/z 948.3.
formed on the fragment ion at m/z 472.2 displayed low
intensity fragments that gave the internal sequence R9T10 V11
D12 [Scheme 3(b)]. Attribution of the arginine as the last
amino acid of the fragment m/z 472.2 was deduced by a cal-
culation based on the mass difference between the precursor
ion and the identified sequence tag T10 V11 D12 [472.2 –
101(T) – 99(V) – 115(D) = 157.2 which corresponds to
(R+H)
+]. Tryptic digestion of DIM13 and analysis by
MALDI-MS confirmed this attribution.
Characterization of the fragment at m/z 472.2 allowed
the interpretation of minor ions obtained from the MS3of the
(2+) precursor ion at m/z 732.3 which were attributed as inter-
nal fragments containing the sequence R9T10 V11 D12 [Table 1
and Scheme 3(a)]. Moreover, the (2+) fragment ion at m/z
603.2 identified as H3V4E5R6P7D8R9T10 V11 D12, confirmed
the presence of H3on the N-terminal side of the DIM13 pep-
tide.The last N-terminal amino acids containing the N-termi-
nal blocking group were deduced from the MS2and MS3
results as follows. The N-terminal blocking group was
assumed to be a pyroglutamic acid (Z in the one-letter code)
as this is frequently observed for small bioactive peptides
and supported by the loss of 112.8 Da from the m/z 846.7
ion. To confirm this result, an MS3experiment was per-
formed on the ion at m/z 846.7. The obtained mass spectrum
displayed ions (Table 1) formerly characterized as y-ions
and internal-fragment ions. Moreover, b-fragment ions
(denoted as b* fragments) corresponding to original b-frag-
ment ions minus the mass of a pyroglutamic residue
(112 Da) were also observed. Therefore, since no b-ions of
the DIM13 peptide were observed, the precursor ion at m/z
846.7 was unequivocally assigned to be DIM13 without its
pyroglutamic N-termination.
Considering the pyroglutamic acid N-termination and
the smallest b-ion (b3at m/z 396.2) detected in the fragmen-
tation spectra, we could identify the amino acid located at the
second position of the N-terminal side. Thus, the b3-ion at
m/z 396.2 was shown to contain a histidine residue (137 Da)
plus the pyroglutamic acid N-termination (112 Da), so that a
phenylalanine (147 Da) was deduced as the missing amino
acid on the N-terminal side of DIM13 peptide. To confirm
the presence of a phenylalanine at the second position, the
b3-ion at 396.2 m/z was further fragmented (MS4). The corre-
sponding MS4spectrum exhibited one major fragment at m/z
241.7, which justified the attribution of the phenylalanine.
The C-terminal sequence of DIM13 was elucidated
using the MS3fragmentation from the (1+) precursor ion at
m/z 1187.6 [Figure 4(c) and Scheme 3(c)]. The MS3spectra
displayed series of y ions (Table 1) and identified the C ter-
minus as F13 G14 N15 G16 G17 F18 S19 A20 Q/K21 R22 F23 [Scheme
3(c)]. All the interpretations from the MS2,MS
3and MS4
analyses led to the following DIM13 sequence:
Z1F2H3V4E5R6P7D8R9T10 V11 D12 F13 G14 N15 G16 G17 F18 S19
A20 Q/K21 R22 F23
In our sequence determination, we still had an ambigu-
ity for the attribution of the residue at position 21, which
could be either a glutamine or a lysine amino acid. These
isobaric residues can be differentiated under high-energy
collision experiments, thanks to the formation of a specific
d-fragment ion.15 More recently, Bahr et al.16 have described
a methodology, based on MSnexperiments performed using
an ion trap, which allows the differentiation of glutamine
from lysine. By sequential MSnexperiments on b- or y-frag-
ment ions adjacent to the ambiguous residue, a peptide tag
containing a glutamine residue loses a formamide molecule
(45 Da) from the lateral chain, which is specific enough to
differentiate the two isobaric residues.
Accordingly, we attempted an MS3experiment on the
y3-ion at m/z 450.3, which is a fragment ion adjacent to the
ambiguous residue. However, the spectrum obtained did not
allow a determination of the residue at position 21 as
described by Bahr et al.16 Therefore, to elucidate the nature
of the ambiguous residue at position 21, trypsinolysis of the
HPLC fraction containing DIM13 with other co-eluted pep-
tides, was performed. At this stage of the work, the sequence
of DIM13 was known and it was, therefore, possible to
clearly identify the DIM13 tryptic peptides among the oth-
ers. The enzymatic digest analyzed by MALDI-MS notably
showed the following identified ions for the DIM13 peptide:
m/z 1166.2 (Z1F2H3V4E5R6P7D8R9), m/z 1355.6 (T10 V11 D12
F13 G14 N15 G16 G17 F18 S19 A20 Q/K21 R22) and m/z 1503.7 (T10 V11
D12 F13 G14 N15 G16 G17 F18 S19 A20 Q/K21 R22 F23). A lysine in
position 21 would have led to tryptic fragments at m/z
2348.5, m/z 1568.6 and m/z 1200.3. These results allowed us
to identify a glutamine residue at position 21, if no missed
cleavages occurred.
The elucidation of the primary structure of DIM13 was
complex since MS2experiments generated only a few frag-
ments and the use of MSn(n> 2) was therefore necessary to
recover the whole sequence. The fragmentation behavior of
the DIM13 peptide is considerably different from that of
tryptic peptides which end with an arginine or a lysine and
which are known to fragment easily thanks to the permanent
charge located on the C-terminus.17 As the DIM13 peptide
contains 23 amino acids, high charge states (4+and 3+) are
required to have enough energy to fragment this large pep-
tide. Fragmentation of high-charge-state ions generates
daughter ions of the same charge state, or lower, which has
to be precisely determined to interpret the mass spectra. The
ion trap analyzer offers sufficient resolution to distinguish
the different charge states and was therefore appropriate to
sequence 4+and 3+ions for the characterization of the
DIM13 peptide.
From the interpretation of all the MS2mass spectra, 38%
of the DIM13 sequence was elucidated. The data from MS3
and MS4spectra, performed using chosen fragment-ion pre-
cursors characterized the entire primary structure of this
peptide. Nevertheless, the few fragments obtained by MS2
for this peptide correlate with previous studies showing that
the observed fragmentations depend on the nature and
charge state of the precursor ion of the peptide.18 Confirma-
tion using model peptides showed that the number of ioniz-
N. Carte et al.,Eur.J. Mass Spectrom.7, 399–408 (2001) 407
ing protons relative to the number of basic residues present
in peptides containing aspartic acid (D) or glutamic acid (E)
residues influences the dissociation patterns of protonated
peptides. Specific and dominant cleavages at acid residues
were shown to occur when the number of ionizing protons
equals the number of arginine residues in these peptides. On
the contrary, when the number of ionizing protons is greater
than the number of arginine residues, non-specific cleavages
are observed.
According to previous observations, the MS2experi-
ments of the (3+) molecular ion (all the ionizing protons are
located on the three arginine residues) exclusively displayed
series of b and y ions adjacent to the acidic amino acid resi-
dues at m/z 624.2 (1+)b
5,m/z 992.3 (1+)b
8,m/z 732.3 (2+)b
12
and m/z 829.7 (2+)y
15,m/z 1187.6 (1+)y
11 as major fragments
[Scheme 1(b)]. On the other hand, MS2fragmentation per-
formed on the (4+) molecular ion (the ionizing protons are
now more numerous than the number of arginine residues)
exhibited a more complex tandem mass spectrum with non-
specific fragmentations that was, nevertheless, richer in
sequence information [Scheme 1(a)].
Conclusions
This work shows that the complete sequence determina-
tion of a novel natural 23-amino-acid peptide with a blocked
N-terminus may be achieved by a combination of MSnanaly-
ses performed on an ion trap analyzer, MALDI-MS with
enzymatic digests and the use of a fast HPLC procedure. It is
significant to point out that MS3and MS4experiments pro-
vided key sequence information whereas MS2experiments
yielded only a small amount of sequence information.
Finally, using this structural information, searches in
the Berkeley Drosophila Genome Project data base (BDGP)
using the TBLASTN program now found two nucleotide
sequences encoding DIM13 which were located on the same
chromosome arm (2R) in 50A9–50A9 according to the fix
annotations (CG18278, CG18279) from the Genome Anno-
tation Database of Drosophila (GadFly). The sequence of the
DIM13 peptide has been patented on 29 November 2000
under the number 00/15434. The total identification of the
two nucleotide sequences suggests a gene duplication. In
addition, in the vicinity of DIM13, two additional isoforms
were observed with 81% and 68% of amino acid sequence
similarities.
Acknowledgement
This work was supported by grants from CNRS
(Programme Biologie Cellulaire), Association Française de
Lutte contre la Mucoviscidose (AFLM) and NIH (1PO1
AI44220-02). N.C. thanks the Bruker Daltonics Society and
the CNRS for financially co-supporting her PhD fellowship.
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Received: 5 September 2000
Accepted: 24 March 2001
Web Publication: 5 October 2001
408 De Novo Sequencing of an Immune-Induced Peptide of Drosophila Melanogaster
