Spatio-temporal dynamics of the egg-laying-inducing peptides during an egg-laying cycle: a semiquantitative matrix-assisted laser desorption/ionization mass spectrometry approach

Spatio-temporal dynamics of the egg-laying-inducing peptides

during an egg-laying cycle: a semiquantitative matrix-assisted laser

desorption/ionization mass spectrometry approach

C. R. Jimenez,* A. ter Maat,� A. Pieneman,� A. L. Burlingame,� A. B. Smit* and K. W. Li*

*Department of Molecular and Cellular Neurobiology and �Department of Developmental and Behavioral Neurobiology,

Faculty of Earth and Life Sciences, Vrije Universiteit, Amsterdam, the Netherlands

�Department of Pharmaceutical Chemistry, Mass Spectrometry Facility, University of California San Francisco, San Francisco,

California, USA

Abstract

The activity-dependent release of peptides from the neuro-

endocrine caudodorsal cell (CDC) system of the freshwater

snail Lymnaea stagnalis regulates egg laying and related

behaviors. In this study, we optimized a mass spectrometry-

based approach to study the spatio-temporal dynamics of

peptides that are largely derived from the CDC hormone

precursor during an egg-laying cycle and a CDC discharge

in vitro. Semi-quantitative peptide mass profiling using matrix-

assisted laser desorption/ionization mass spectrometry

(MALDI-MS) indicated a massive depletion of peptides from

the neurohemal area in the cerebral commissure (COM)

during egg laying and the existence of a reserve pool of

peptides in the CDC somata that were transported to the COM

to restore peptide levels. The depletion of CDC peptides from

the COM was correlated to their release during an induced

electrical discharge in vitro. Moreover, MALDI-MS of the rel-

easate revealed extensive truncation of the carboxyl terminal

peptide. Finally, two novel peptides of 1788 and 5895 Da, not

encoded by the CDC hormone precursor, also exhibited

temporal quantitative changes similar to those of CDC pep-

tides. Sequencing of the peptide of 1788 Da by tandem mass

spectrometry yielded the novel sequence HF(FH)FY-

GPYDVFQRDVamide. Together, this implicates a more

complex set of CDC peptides for the regulation of egg laying

than previously anticipated.

Keywords: mass spectrometry, neuropeptide, pond snail,

releasate, reproduction.

J. Neurochem. (2004) 89, 865–875.

Neuro-endocrine peptides form a structurally diverse class of

signaling molecules and play important roles in animal

physiology and the organization of behavior (Li 2001). It is

often suggested that multiple peptides are needed to coordi-

nate behavior in well-defined temporal patterns; however, the

complexity of most vertebrate neuronal systems poses

considerable difficulty for the detailed analysis of the

functional contribution of peptide cocktails. During the past

decades, several invertebrate models have been developed to

understand the physiological role of distinct peptides

contained in single/multiple precursors (Nassel 2002; El

Filali et al. 2003; Orekhova et al. 2003). Neuro-endocrine

peptidergic systems controlling egg laying in molluscs have

been particularly well studied (Geraerts et al. 1991; Wayne

et al. 2004) and have revealed novel principles concerning

cotransmission (Brussaard et al. 1990; Jung and Scheller

1991; Klumperman et al. 1996). In the freshwater snail

Lymnaea stagnalis egg laying is initiated by a group of

command neurons, the caudodorsal cell (CDC) neurons, in

the CNS (Geraerts et al. 1991). These neurons express a

gene encoding the CDC hormone (CDCH) precursor that

contains multiple peptide domains (Vreugdenhil et al. 1988;

Fig. 1). Interestingly, the distinct peptides generated from

different parts of the CDCH precursor exhibit differences in

Received August 24, 2003; revised manuscript received November 28,

2003; accepted December 19, 2003.

Address correspondence and reprint requests to Dr C. R. Jimenez,

Department of Molecular & Cellular Neurobiology, Research Institute

Neurosciences, Vrije Universiteit, De Boelelaan 1085, 1081 HV,

Amsterdam, the Netherlands. E-mail: [email protected]

Abbreviations used: CDC, caudodorsal cell; CDCH, CDC hormone;

CDCP, CDC peptide; COM, cerebral commissure; CTP, carboxyl ter-

minal peptide; DMSO, dimethylsulfoxide; MALDI-MS, matrix-assisted

laser desorption/ionization mass spectrometry.

Journal of Neurochemistry, 2004, 89, 865–875 doi:10.1111/j.1471-4159.2004.02353.x

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 865–875 865

stoichiometry (Li et al. 1994), suggesting distinct sorting and

processing events for these peptides (Klumperman et al.

1996). Physiological experiments indicated that peptides

derived from the amino terminal region of the precursor are

involved in the modulation of neuronal activity in the CNS

during the initiation of egg laying (Brussaard et al. 1990),

whereas those located at the carboxyl region, such as the egg-

laying hormone CDCH, may also function as a hormone with

a long-range effect on the peripheral organs (Ebberink et al.

1985). As the differential storage and temporal release of the

peptides may underlie the overall physiological effects of the

peptides derived from this single precursor, it is important to

follow their dynamics during an egg-laying cycle. Egg laying

in Lymnaea takes place about once every 3–4 days. The

onset of the whole egg-laying processes is signaled by a long

electrical discharge of CDCs that lasts for about 60 min

(reviewed by Geraerts et al. 1991 and Ter Maat 1992).

During this time the CDC peptides are released from the

neurohemal area of CDCs, the cerebral commissure (COM).

In the following 2–3 h the covert (ovulation of oocytes,

formation of the eggs and the egg mass) and overt behaviors

(resting, turning and egg mass deposition) take place. In this

study, we used semiquantitative matrix-assisted laser desorp-

tion/ionization mass spectrometry (MALDI-MS) (cf. Jimenez

et al. 1997) to analyze distinct anatomical compartments of

the CDC system, i.e. the CDC somata and axon terminals in

the COM during egg laying in vivo as well as in the COM

and releasate during an in vitro discharge. This MS-based

peptidomics approach allowed us firstly to unequivocally

demonstrate massive depletion of CDC peptides from the

COM during egg laying and secondly to relate this depletion

to release of CDC peptides. Moreover, the relatively slow

depletion (only between 10 and 24 h) of CDC peptides from

the CDC somata could be related to a slow replenishment

(> 24 h) of the axon terminals in the COM, which may play

a role in the timing of the egg-laying cycle in these animals

(egg laying occurring every 2–3 days). Finally, the analysis

of the peptide profile of the in vitro releasates has yielded

insights into the fate of peptides after their release and may

underscore the possible role of peptides as a transmitter [i.e.

a CDC peptide (CDCP), carboxyl terminal peptide (CTP)

and d peptide] and/or a hormone (i.e. CDCH). Moreover, the

quantitative changes in the level of two novel peptides of

5895 and 1788 Da during egg laying and a CDC discharge

indicated that these peptides may also be involved in the

control of egg laying. Tandem mass spectrometry revealed

the novel structure of the 1788-Da peptide as HF(FH)FY-

GPYDVFQRDVamide.

Materials and methods

Egg-laying experiment

Adult specimens of L. stagnalis (shell height 30–35 mm), bred

under standard laboratory conditions (20�C, 12 h light/12 h dark

cycle, lettuce ad libitum), were used. Egg laying was induced by the

so-called clean water stimulus (Ter Maat et al. 1983). In short,

animals were kept individually in stagnant water for 5 days at 20�Cand fed lettuce ad libitum. During this period the water polluted and,

as a consequence, the animals ceased egg laying. By placing the

animals in clean aerated water the CDC system was induced to

become active and to release the egg-laying peptides. As a result,

ovulation and egg mass production were initiated.

Animals were killed at various time points (0 min, 45 min, 2 h,

4 h, 10 h and 24 h) after placing them in clean water. At each time

point, animals were killed and the CDCs and COM were dissected.

Material from 15 animals was pooled per sample and at least three

independent samples were obtained per time point. Snails were

checked for the presence of oocytes, eggs or an egg mass in the female

tract to confirm the activation of the CDCs. About 50% of the animals

responded to the clean water stimulus. At 45 min, responding animals

comprised a heterogeneous group, some snails had ovulated whereas

others had already proceeded with their eggs packaged into an egg

mass. These snails were classified separately as 45 min (ovulated)

and 45 min (packaged), respectively. At the later time points the

presence or absence of an egg mass was used to classify the animals.

The data were tested using a single classification analysis of

variance followed by Dunnett’s test for comparison with a control

group. Samples from animals that did not respond to the stimulus to

elicit egg laying were obtained at all time points (0 min, 45 min,

2 h, 4 h, 10 h and 24 h). The levels of CDC peptides from both the

cell bodies and neurohemal area (COM) were not different between

the time points. Therefore, these data were considered to constitute a

single control group against which the peptide levels in responding

animals were tested. JMP software (SAS Institute, Cary, NC, USA)

was used for this analysis.

In vitro release experiment

The experiment was performed as described by Moed et al. (1989).

In brief, cerebral ganglia were dissected and collected on ice in

HEPES-buffered saline containing (in mM): NaCl, 30; NaCH3SO4,

10; NaHCO3, 5; KCl, 1.7; CaCl2, 4; MgCl2, 1.5; HEPES, 10;

pH 7.8 adjusted with NaOH. The stable cAMP analog 8-CPT-cAMP

(Boehringer Mannheim, Mannheim, Germany) was kept as a 10)1M

stock solution in dimethylsulfoxide (DMSO). The releasates were

collected from three independent pools of 15 cerebral ganglia. At

30 min prior to application of 8-CPT-cAMP, the samples were

transferred to room temperature. After this 30-min pre-incubation

period, the medium was removed and exchanged for saline

Fig. 1 The organization of the caudodorsal cell (CDC) hormone

(CDCH) precursor. The precursor contains multiple peptide domains

that are flanked with dibasic processing sites as indicated by black

vertical lines. The tetrabasic site divides the CDCH precursor into the

amino and carboxyl regions and is indicated with a thick black vertical

line and an arrow. sp, signal peptide; Nt, amino terminal peptide; e, e

peptide; b, b peptide; CaFl, calfluxin; a, aCDC peptide; c, c peptide; d,

d peptide; CTP, carboxyl terminal peptide.

866 C. R. Jimenez et al.

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 865–875

containing 10)3M 8-CPT-cAMP and 1% DMSO in the experimental

group or saline containing only 1% DMSO in the control group.

After 15 min, the release media of all experimental groups were

collected and exchanged for fresh saline with 10)3M 8-CPT-cAMP

and 1% DMSO for a second incubation interval of 15 min. Similarly,

after 15 min the release media of the control groups were collected

and exchanged for fresh saline containing 1% DMSO only. The

release media of the experimental and control groups of the

remaining intervals (30–45, 45–60 and 60–90 min) were collected

and each time exchanged for fresh saline alone. At each time interval,

release medium was collected from the pools of cerebral ganglia (15

per incubation). Each sample was measured twice, yielding six

measurements per time interval. All experiments were carried out in

the absence of protease inhibitors. All collected media were acidified

and immediately frozen. After the last incubation interval the COMs

were dissected and immediately frozen.

Extraction and pre-purification of peptides

Dissected entire clusters of CDCs, COMs and release media were

separately collected on solid carbon dioxide and stored at ) 55�C.

The release media, clusters of CDCs and COMs of 15 animals per

group were extracted by boiling in 0.1 M acetic acid for 8 min and

centrifuged for 5 min at 4�C. The supernatant fluids were separately

loaded into a C18 solid phase extraction column (column volume

300 lL; Supelco, Bellefonte, PA, USA). The bound material was

eluted with 200 lL 7.5 mM trifluoroacetic acid in 80% acetonitrile,

followed by 200 lL methanol. The eluates were collected in the

same vial, lyophilized to a volume of 75 lL and then diluted to

150 lL using 0.1% trifluoroacetic acid.

Mass spectrometry

The MALDI-MS measurements of single neurons and the semi-

quantitative analysis of profiles of CDC peptides were performed

essentially as previously described (Jimenez et al. 1994, 1997). For

the direct analysis of cells and tissue (Jimenez et al. 1994),

individual CDC soma and biopsies of COM were dissected under

a microscope and directly transferred to 0.7 lL matrix using a fine

glass pipette (tip diameter �30 lm) and a pair of fine forceps,

respectively. For quantitation, MALDI-MS was performed on C18

pre-purified extracts (see below). From each of the pre-purified

extracts of the CDCs, COMs and releasates, 0.7 lL (representing

7% of one animal equivalent) was mixed with 1 lL of matrix

(10 mg 2,5-dihydroxy-benzoic acid and 1 mg 5-methoxy-2-benzoic

acid dissolved in 1 mL 7.5 mM trifluoroacetic acid in 30%

acetonitrile) containing a fixed amount of reference peptide (see

below) on a stainless steel target. The samples were dried by a

stream of cool air and analyzed using a laboratory-built laser

desorption reflectron time-of-flight mass spectrometer equipped with

a pulsed nitrogen laser (337 nm; pulse width 3 ns). Measurements

of the extracts of CDCs and COMs of the egg-laying experiment and

the COMs and release media of the release experiment were carried

out at acceleration potentials of 4.4 and 3.4 kV, respectively. Internal

calibrations were performed on identified molecular ions (i.e.

peptides derived from the CDCH precursor), yielding an accuracy of

mass measurement in the range of 0.1%.

For quantitation, peptide signals were quantified relative to an

internal reference peptide that was incorporated in the matrix and

two independent measurements were performed by scanning around

the crystal rim of the matrix/analyte preparation. The internal

reference peptide, rat b-endorphin, was added to the matrix at a

concentration of 0.5 pmol/lL for measurement of the CDCs and

COMs and 0.25 pmol/lL for measurements of the release media. To

assess the MALDI-MS quantification method, a dilution series of a

COM extract was analyzed by MALDI-MS using a fixed internal

reference peptide concentration of 0.5 pmol/lL. The quantified ion

signals of the CDC peptides in the COM dilution series (n ¼ 3 per

dilution) were fitted using linear regression (Sigma Plot software,

Rockware Inc., Golden, CO, USA).

For peptide sequencing, high-energy collision-induced dissoci-

ation tandem mass spectrometry of the peptide at m/z 1789 from

COM extract was performed using an Autospec-TOF equipped with

a MALDI source as previously described (Medzirhadszky et al.

1996; Li et al. 1997). The sample was prepared as stated above.

Results

Mass spectrometric peptide profiling of caudodorsal cell

somata and cerebral commissure

The mass spectra of the CDCs (Fig. 2a) and COM (Fig. 2b)

contained molecular ions corresponding to the protonated

masses of bCDCPs, aCDCP, calfluxin, d peptide, CTP and

CDCH. These peptides are all derived from the CDCH

precursor (Fig. 1) that is specifically expressed in the CDCs.

The mass spectra also revealed different stoichiometries for

the two sets of peptides derived from either the amino

terminal region or the carboxyl terminal region of the CDCH

precursor, with the carboxyl region peptides, i.e. the CTP,

CDCH and d peptide, being far more abundant than the

amino region peptides in both the CDCs and the COM. To

exclude the possibility that the differences in stoichiometries

of the peptides detected by MALDI-MS are a technical bias

caused by differences in ionization and/or detection efficien-

cies of the peptides, we mixed equal amounts of synthetic

peptide, i.e. b1 peptide, b3 peptide, d peptide, CTP and

CDCH, and then quantified their ion signals. Figure 2(c)

shows that the ion intensities of the tested amino terminal

CDC precursor peptides, b1 and b3, were similar to the tested

carboxy terminal CDC precursor peptides, d peptide, CTP

and CDCH. Therefore, differences in ionization between the

amino terminal and carboxy terminal CDC precursor peptides

can probably not explain the observed different stoichiome-

tries between the ions in the CDC and COM. Moreover, the

difference in stoichiometries of CDC peptides observed in the

CDCs and COM is in agreement with the results from

previous reports employing alternative methodologies, i.e.

immunocytochemistry (Van Heumen and Roubos 1991) and

conventional peptide chemistry (Li et al. 1994), and has been

considered as evidence for differential processing of distinct

peptides derived from a single precursor.

While the ion signals of the peptides derived from the

carboxyl region of the precursor are intense and can easily

Peptidomics of egg laying in Lymnaea 867


be quantified in relative terms by comparison with an

internal reference standard, many of the peptides derived

from the amino region yield weak signals. Of several amino

region-derived peptides, aCDCP consistently yielded a

signal several fold above background and so could be

quantitatively detected. Therefore, this single peptide was

quantified as a representative of the amino terminal-located

peptides.

Matrix-assisted laser desorption/ionization mass

spectrometry quantitation of caudodorsal cell peptides

The direct analysis of single cells and tissues requires the

disruption of the samples in the matrix and the passive

uncontrollable leakage of peptides to the matrix. For

quantitation purposes, however, all of the samples should

behave in a reproducible manner. To this end, the peptides in

the CDCs and the COMs were separately extracted by

boiling in acetic acid and then pre-purified by a C18 solid

phase extraction column. Figure 3 shows that the mass

spectra of CDC and COM extracts are similar to those of the

direct analysis of CDC and COM (Fig. 2). Moreover, the ion

intensities from different COM extracts are similar.

In order to determine whether the changes in mass

spectrometric ion signal levels as quantified relative to the

internal reference peptide approximately follow the actual

changes in levels of the CDC peptides, a dilution series of a

COM extract was analyzed by MALDI-MS using a fixed

internal reference peptide concentration of 0.5 pmol/lL.

Figure 4 shows that all quantified ion signals of the two

smaller peptides, aCDCP (1167.6 Da) and d peptide

(1565.7 Da), of the COM dilution series could be fitted

using a linear regression model with regression coefficients

of 0.995 and 0.994, respectively. The linear dynamic range of

the two larger peptides, CTP (2588.2 Da) and CDCH

(4473.4 Da), was more restricted with the signal of the

(a)

(b)

(c)

Fig. 2 Matrix-assisted laser desorption/ionization mass spectrometry

(MALDI-MS) analysis of peptide profiles in the caudodorsal cell (CDC)

system. Direct MALDI-MS analyses of (a) freshly dissected single

CDC and (b) biopsies of the cerebral commissure. Peptides with

masses corresponding to the CDC peptides, b peptides (b), aCDC

peptide (CDCP) (a), d peptide (d), carboxyl terminal peptide (CTP),

calfluxin (CaFl) and CDC hormone (CDCH) are indicated. aCDCP is

present in the spectra both as a protonated ion and a cationized ion.

Molecular ions of unidentified molecular species are labeled with their

mass. (c) Quantification of synthetic CDC peptides loaded in equal

ratio on the MALDI target. A peptide mixture containing 0.5 pmol each

of synthetic b1 and b3 peptide, d peptide, CTP and CDCH was loaded

in six spots to the target and analyzed by the mass spectrometer. Ion

peaks of each peptide were quantified by six different measurements.

X-axis, m/z, mass : charge ratio; Y-axis, arbitrary units. e, e peptide.

Rel

ativ

eIn

tens

ity

Rel

ativ

eIn

tens

ity

1000

(b)

(a)

2000 4000 6000

α

CTPCDCH

5895.9

CTP

CDCH

5896.81790.3α

δ

δ

M/Z

1000 2000 4000 6000M/Z

616.1

1788.9

2238.0 3136.72735.2

2238.3

Fig. 3 Examples of mass spectra obtained after a peptide extraction

from (a) clusters of caudodorsal cell and (b) cerebral commissure

dissected and pooled from 15 animals that had not laid eggs for

3 days. See Fig. 2 for peak labels.



undiluted sample being relatively low. When the data point

of the undiluted sample (dilution quotient 1) was excluded,

the ion signals of CTP and CDCH could be fitted using linear

regression, yielding regression coefficients of 0.968 and

0.969, respectively (Figs 4c and d). Therefore, we used a

twofold diluted sample for quantification of the ion signals

during egg laying. As previous experiments indicated that

CDC peptide levels during the whole egg-laying process are

reduced about twofold (Vreugdenhil et al. 1985), the

MALDI-MS quantitation of a twofold diluted sample is

adequate.

Changes in the peptide levels in caudodorsal cells and

cerebral commissure during an egg-laying cycle

Clean water stimulation is known to induce a sustained

electrical discharge in CDCs, which is thought to lead to

peptide release in the COM. Mass spectrometric analyses

revealed that the clean water stimulus induced distinctly

different patterns of changes in ion signal levels of peptides

in the COM and the CDCs. The peptide levels in the COM of

the animals that had just ovulated did not change signifi-

cantly (Fig. 5). However, in the group that showed pack-

aging of the eggs, a 40–50% reduction occurred in the levels

of the quantified CDC peptides. At 2 h after the clean water

stimulus, around egg mass deposition, a further slight

reduction of peptide levels seemed to occur. In contrast to

that of the COM, the ion signal levels of the peptides in the

CDCs remained unchanged up to 10 h after the clean water

stimulus. At 24 h after the clean water stimulus, the peptide

levels in the CDCs decreased by 55–70%. This reduction of

CDC peptides correlates to the slight increase of peptides in

the COM, suggesting that the peptides were transported from

the CDCs to the COM.

Changes in the peptide levels in releasates and in cerebral

commissure during an 8-CPT-cyclic AMP-induced

electrical discharge of the caudodorsal cell system kept

in vitro

As the analysis of the CDC system during an egg-laying

cycle in vivo showed specifically that the peptide levels in the

COM decreased during egg mass production, we assumed

that this was caused by a massive release of peptides from the

neurohemal CDC axon endings in the periphery of the COM.

To examine peptide release during an induced discharge of

the CDC system, 8-CPT-cAMP was applied in vitro to

dissected cerebral ganglia connected by the COM. The

application of 8-CPT-cAMP induces an electrical discharge

in the CDCs within 2.5 min and, therefore, the induction of

peptide release in vitro is relatively fast.

Figure 6 shows examples of the MALDI-MS peptide

profiles in the release media before () 30 to 0 min) and after

(0–15 min) the onset of the 8-CPT-cAMP-induced electrical

CDC discharge. During the pre-incubation period of 30 min

only trace amounts of CDC peptide ions could be detected

(Fig. 6a). After stimulation with 8-CPT-cAMP, the CDC

peptides were detected in the releasates (Fig. 6b). The

peptide profiles of 8-CPT-cAMP releasates were similar to

the profiles of releasates collected after electrical induction of

the CDC discharge (data not shown). Furthermore, the

difference in the stoichiometry of the amino region and

carboxyl region peptides of the CDCH precursor as demon-

strated in the COM is also found in the releasate (Figs 6 and

7). The cumulative curves of Fig. 7(b) reveal that the release

rates of CDC peptides were highest in the second interval

(15–30 min). In addition to the intact peptides, molecular ion

species with masses corresponding to the truncated forms of

CTP (residues 4–22 and 5–22) and d peptide (residue 9–15)

were clearly detected in the releasates. These truncated

peptides were already present in the releasates collected in

the first interval (Figs 7c–d). At later intervals (15–90 min)

these truncated forms became progressively more abundant.

Truncated forms of CDCH (Fig. 7e) were also detected,

Fig. 4 Average caudodorsal cell (CDC) peptide levels quantified rel-

ative to the internal standard (rat b-endorphin) in a dilution series of a

cerebral commissure (COM) extract. Average values of triple matrix-

assisted laser desorption/ionization mass spectrometry measure-

ments of the same COM extract are plotted. Error bars indicate SDs.

The relative amount was determined by dividing the peak height of the

peptide by the peak height of the standard. Regression coefficients of

linear regression (solid line) through the quantified ion signals are

0.995 [aCDC peptide (CDCP)] (a), 0.994 (d peptide) (b), 0.968

[carboxyl terminal peptide (CTP)] (c) and 0.969 [CDC hormone

(CDCH)] (d) (with omission of the highest data point obtained for CTP

and CDCH). Solid line, linear regression. X-axis, the reciprocal of the

dilution factor (undiluted/diluted); Y-axis, relative amount.



albeit at a lower level. At the end of the whole incubation

period, i.e. after 90 min, an �50% reduction in the ion signal

level of the peptides in the COM was apparent (Fig. 7a),

which is very similar to the degree of depletion seen after egg

laying (cf. Fig. 5).

Semi-quantitative MALDI-MS analysis requires summa-

tion of signals from many matrix crystals of variable quality.

This approach may be less sensitive than qualitative MALDI-

MS analysis, which focuses only on a single or at the most a

few crystal spots that give a high signal-to-noise ratio. As

such, it is likely that some of the minor (putative) truncated

forms of CDC peptides may not have been detected by

semiquantitative MALDI-MS analysis. We, therefore, also

performed qualitative MALDI-MS analysis to gain a better

insight into the patterns of truncation of the released CDC

peptides. Figure 8(a) reveals the presence of a large number

of (putatively) truncated forms of CTP (1–11), d peptide

(9–15) and CDCH (2–36, 3–36, 4–36) from the release

medium collected during 45–60 min after the onset of

8-CPT-cAMP stimulation. Figures 8(b–d) schematically

indicate the cleavage sites of CTP, d peptide and CDCH,

respectively. The CTP was predominantly cleaved at the

Ala–Phe bond (yielding CTP4)22), which was followed by

the removal of Phe (yielding CTP5)22). The carboxyl

terminal Phe residue of CTP was removed to a much lesser

extent. In addition, a series of amino terminal truncated CTP

and a specific cleavage at the carboxyl terminal at the Asp–

Tyr bond were found at lower levels. The truncated d peptide

could be formed by the cleavage at the Ser–Ala bond,

whereas the truncation of CDCH involved primarily the

removal of the amino acids at the amino terminus, i.e. the

sequential processing of Leu, Ser and Ile. Qualitative

MALDI-MS of COM tissue never revealed the above

observed truncated CDC peptides (Fig. 8e), indicating that

the peptides are specifically cleaved after their release.

Evidence of novel caudodorsal cell cotransmitters

Mass spectrometric analyses of CDC and COM revealed the

presence of molecular ions that do not correspond to peptides

that may be derived from the CDCH precursor, e.g. the

molecular ions at m/z 1789 and 5896 (Figs 2 and 3),

Fig. 5 Average peptide levels in the caudodorsal cells (CDC) and the

cerebral commissure (COM) during an egg-laying cycle of Lymnaea.

The peptide levels in the CDCs (left panel) remained about constant

during egg mass production and afterwards but had significantly de-

creased at 24 h after the clean water stimulus. In the COM (right

panel) most of the changes in peptide levels occurred during egg mass

production. Shortly after ovulation, no changes in peptide levels were

observed but, during packaging of the eggs, the levels of CDC hor-

mone (CDCH), d peptide, aCDC peptide (CDCP) and two novel pep-

tides detected at m/z 1789 and 5896 were significantly reduced by

�40–50%. The peptide levels in the COM remained low and only

tended to increase again at 24 h after the clean water stimulus. The

shaded bar denotes the pooled control group of the animals that did

not respond to the stimulus to elicit egg laying and were obtained at all

time points (0 min, 45 min, 2 h, 4 h, 10 h and 24 h). For each time

point, the average value of at least six measurements on three sam-

ples (15 animals pooled per sample) is plotted [control, n ¼ 16; ovu-

lation (OV), n ¼ 3; packaging of the eggs (PA), n ¼ 3; egg laying (EL)

120 min, n ¼ 6; EL 240 min, n ¼ 3; EL 600 min, n ¼ 3; EL 1440 min,

n ¼ 3]. Error bars indicate SDs. *Statistically significant (Dunnett’s

test). X-axis, the time points after the snails were transferred to the

clean water condition. Y-axis, relative amount of the signal intensities

of the peptides compared with the internal standard. CTP, carboxyl

terminal peptide.

OV PA EL

CTP

δ peptide

αCDCP

17881788

5896

αCDCP

Rel

ativ

e am

ount

CDCH

CDC somata Commissures

OV PA EL

δ peptide

CTP

CDCH

0 45 45 120 240 600 1440

0 45 45 120 240 600 1440

0 45 45 120 240 600 1440

0 45 45 120 240 600 1440 0 45 45 120 240 600 1440

0 45 45 120 240 600 1440

0 45 45 120 240 600 1440

0 45 45 120 240 600 1440

0 45 45 120 240 600 1440

0 45 45 120 240 600 1440

0 45 45 120 240 600 1440

0 45 45 120 240 600 1440

*

*

*

* * * **

* *

* *

** * *

* * *

* ***

* * * *

0.08

0.16

0.24

0.32

0.4

0.2

0.32

0.16

1.6

0.8

2.4

1.0

0.5

2.4

1.2

Time (min)

5896

*

0

1.5

1.0

0.5

0

1.2

1.8

0.6

0

0.8

1.2

0.4

0

0.50

0.25

0.75

0

0.8

1.2

0.4

0

0.14

0.07

0.21



suggesting that they may represent novel CDC peptides.

These peptides were both depleted from the COM during egg

laying in vivo and a cAMP-induced discharge in vitro (Figs 5

and 7a). Moreover, the 5895-Da peptide was detected in the

releasates (Fig. 6b) and followed release dynamics similar to

other CDC peptides (Fig. 7a), indicating that this novel

peptide also displays activity-dependent release from the

COM. However, the 1788-Da peptide could not be detected

in the releasates, suggesting that this peptide is susceptible to

extracellular protease activity and degraded more rapidly

than the other CDC peptides.

Structural characterization of the novel peptide

To establish the structures of the molecular ions at m/z 1789

and 5896, we subjected them to high-energy collision tandem

mass spectrometry. No useful informative sequence ion was

obtained from the 5895-Da species, which is not unexpected

because it is generally difficult to fragment peptides of high

molecular weight. The MS/MS spectrum of the m/z 1789 ion

contained information that reveals the amino acid composition

as well as the peptide sequence (Fig. 9). The occurrence of

immonium ions at m/z 70.0, 72.0, 84.0, 88.0, 110.1, 120.0,

129.1, 136.1 and 166.3 indicates the presence of the amino

.

CTP 4-22CTP 5-22

CTP

0.2

0.4

Time (min)

Rel

ativ

eA

mou

nt

60 9015 45300

0.8

0.6

CTPαCDCP

CDCHδ

5896

Time (min)

Rel

ativ

eA

mou

nt

60 9015 45300

0.4

0.5

0.1

0.3

0.2

CD

CH

CDCHCDCH 2-36

Time (min)

Rel

ativ

eA

mou

nt

60 9015 45300

60 9015 45300

Time (min)

Rel

ativ

eA

mou

nt

Rel

ativ

e A

mou

nt

3

2.25

0.75

1.5

δ

1789

5896

CT

P

αCD

CP

Control

8-CPT-cAMP

δδ 9-15

Time (min)

Rel

ativ

eA

mou

nt

60 9015 45300

**

*

*

0.8

1.0

0.2

0.6

0.4

0.8

1.0

0.2

0.6

0.4

0.8

1.0

0.2

0.6

0.4

**

(a) (b)

(c) (d)

(e) (f)

Fig. 7 Changes in the peptide levels in cerebral commissure (COM)

and releasates during an 8-CPT-cAMP-induced electrical discharge

in vitro of the caudodorsal cell (CDC) system. (a) 90 min after an in vitro

8-CPT-cAMP-induced discharge the peptide levels in the COM were

significantly depleted by �50% compared with the control COM. (b–f)

Cumulative curves of the peptides in the releasates. (b) The peptide

levels of aCDC peptide (CDCP), d peptide, carboxyl terminal peptide

(CTP) and CDC hormone (CDCH) increased shortly after the onset of

8-CPT-cAMP stimulation, reaching a maximum release rate in the

second interval (15–30 min). (c) Comparison of the increases in the

relative levels of CTP and its amino terminally truncated forms, CTP4)22

and CTP5)22. (d) Comparison of the increases in the relative levels of d

peptide and its amino terminally truncated form, d9–15. (e) Comparison

of the increases in the relative levels of CDCH and its amino terminally

truncated form, CDCH2–36. (f) Cumulative curve of the novel peptide of

5895 Da that was easily detected in the releasates. The novel peptide

of 1788 Da could not be detected in the releasates even though its

depletion from the COM after 8-CPT-cAMP stimulation suggested its

release. For each time point, the average value of six measurements on

three release samples (15 animals per sample) is plotted. Error bars

indicate SDs. The relative amount was determined by dividing the peak

height of the peptide by the peak height of the standard.

Rel

ativ

e In

tens

ityR

elat

ive

Inte

nsity

1000 2000 3000 4000 60005000

1000 2000 3000 4000 60005000

M/Z

CTP

CDCH

5896δ?

αCTP5-22

CTP4-22

Reference

?

CDCH-I 2-36

Reference

-30-0 min

0-15 min

M/Z

(b)

(a)

Fig. 6 Examples of matrix-assisted laser desorption/ionization mass

spectra of in vitro releasates. (a) Control medium collected after a pre-

incubation period of 30 min, prior to the 8-CPT-cAMP stimulation. (b)

Release medium containing 8-CPT-cAMP collected from 0 to 15 min.

Note the presence of amino terminally truncated forms of carboxyl

terminal peptide (CTP) (CTP4)22 and CTP5)22) and caudodorsal cell

hormone (CDCH) (Tr, CDCH2–36). The relative abundances of the

truncated peptides increased in later intervals (see Figs 7 and 8).

X-axis, m/z, mass : charge ratio; Y-axis, arbitrary units.



acid residues Pro, Val, Gln/Lys, Asp, His, Phe, Arg, Tyr and

His, respectively. Using an MS-product listing program with

a parent ion mass of 1789 ± 1.0 Da, the b2, b3, b5, b7, b8,

b9 and b10 fragmentation ions occur at m/z 285.2, 432.3,

652.4, 912.4, 1027.5, 1126.5 and 1273.0. The y ions y3, y4,

y5, y6, y7, y8, y9, y10, y11 and y12 occur at m/z 388.3,

516.4, 663.5, 762.5, 877.5, 1040.6, 1137.6, 1194.6, 1357.7

and 1504.9. There is also a doubly charged species at m/z

569.0 (Y9++) and an internal fragment ion FQ(K)RD at m/z

547.0. The identity of the second residue may be Q or K,

which differ by 0.1 Da and cannot be unequivocally resolved

by this mass spectrometer. In an acetylation experiment, we

established the identity of this amino acid as Q (data not

shown). Together, these fragmentation data yield a sequence

HF(FH)FYGPYDVFQRDVamide. The order of the first two

residues cannot be established because the b1 ion is missing.

This peptide sequence is not present in the EMBL database.

Discussion

We and others have previously reported the qualitative

analysis of released egg-laying peptides (Newcomb and

Scheller 1990) by MALDI-MS in conjunction with nano-

liquid chromatography (Li et al. 1999) and capillary elec-

trophoresis (Rubakhin et al. 2001). However, direct

semiquantitative MALDI-MS has not yet been fully exploi-

ted (see Bucknall et al. 2002 and Jimenez et al. 1997 for

semiquantitative MALDI-MS of biological tissues). In the

present study we first confirmed, using serial dilutions of a

COM extract, that our semiquantitative MALDI-MS analysis

is appropriate for the study of the relative levels of CDC

peptides within the range that they are expected to vary

during an egg-laying cycle. We then applied this methodo-

logy to monitor the relative changes in the ion signal levels

of peptides in the CDCs and the COM during an egg-laying

cycle of Lymnaea in vivo and in the COM and releasates

during an electrical discharge of the CDC system in vitro.

Egg laying of Lymnaea is hallmarked by a massive all-

or-none discharge of all CDCs that leads to the release of a

cocktail of peptides, which contains at least the ovulation

hormone, CDCH, and the excitatory autotransmitter, aCDCP

(Brussaard et al. 1990; Van Heumen and Roubos 1991), that

are both derived from the CDCH precursor. Our results

indicate similar dynamics of release (as demonstrated from

depletion from the COM) of CDC peptides derived from the

.

G S A F F D H I P I I F G E P Q Y D Y Q P FCTP :

δ peptide : S A D S A P S S A N E V Q R F

1 1 0 1 55 2 0

4 - 2 2

5 - 2 2

6 - 2 2

7 - 2 2 4 - 1 3 4 - 1 85 - 1 86 - 1 87 - 1 88 - 1 8

1 0 -1 8

1 - 2 16 - 2 1

9 - 1 5

1 1 0 1 55

CDCH : L S I T N D L R A I A D S Y L Y D Q H K L R E R

Q E E N L R R R F L E L a m i d e

2 - 3 63 - 3 6

4 - 3 6

1 2 34 5

6 7 8

9

1 0

1 1

Rel

ativ

eIn

tens

ity

1000 2000 3000 4000 6000 70005000

CTP

CDCH1-36

5896?

Referenceδ

δ 9-15 2 - 3 6

3 - 3 6

4 - 3 6?

M/Z

?

1000 2000 4000 6000

αCTP

CDCH

5896?

1789.0

M/Z

δ

Reference

Rel

ativ

eIn

tens

ity

??

(a)

(b)

(c)

(d)

(e)

Fig. 8 Matrix-assisted laser desorption/ionization mass spectrometry

(MALDI-MS) analysis displaying proteolytical degradation of carboxyl

terminal peptide (CTP), d peptide and caudodorsal cell (CDC) hor-

mone (CDCH) after release. (a) Mass spectrum of release medium

collected during 45–60 min after the onset of 8-CPT-cAMP stimula-

tion. The spectrum was generated by summation of 150 spectra from a

single matrix crystal that yielded ion signals with high signal-to-noise

ratio. The MALDI-MS profile reveals the presence of many truncated

forms of CTP (1–11) as well as of d peptide (d9–15) and CDCH (2–36,

3–36 and 4–36). X-axis, m/z, mass : charge ratio; Y-axis, ion intensity

in arbitrary units. (b–d) Amino acid sequences of CTP (b), d peptide (c)

and CDCH (d). The sites of cleavage are indicated by vertical lines.

Numbers along the lines indicate the residue numbers of the truncated

fragments observed in the MALDI-MS spectrum of (a). Box numbers

indicate the major truncated peptides in the releasate as detected in

the MALDI-MS spectrum of (a). Numbers above the sequences indi-

cate the residue number. (e) Qualitative MALDI-MS spectrum of cer-

ebral commissure extract. Note the absence of the putatively

truncated CDC peptides. For abbreviations see Fig. 2.



amino and carboxyl termini of the CDCH precursor during

egg laying in vivo. In animals that had just ovulated in

response to the clean water stimulus, no significant reduction

in CDC peptide levels in the COM was apparent, suggesting

a low rate of peptide release. Apparently, the gonad is highly

sensitive to low amounts of circulating ovulation hormone.

Indeed, only 1% of COM extract suffices to induce ovulation

and egg laying (Geraerts et al. 1991). The significant

reduction in CDC peptides in the COM of 40–50% during

packaging of the eggs and 50–70% around oviposition as

determined by quantitative MALDI-MS is in line with

previous findings showing that the ovulation-inducing

potency of the COM extract decreased quickly, by �70%,

during a CDC discharge (Geraerts et al. 1991) and with the

�50% depletion of CDC peptides in the COM at the end of

an 8-CPT-cAMP-induced discharge (this study). The relat-

ively slow depletion of CDC peptides from the CDCs

10–24 h after the induction of egg laying may indicate

transport of the peptides to the COM. However, in this time

period, CDC peptide levels in the COM remained low and

only tended to increase at 24 h. Possibly, this slow replen-

ishment plays a role in the 2–3 day refractory period in

which no egg laying occurs.

Our MALDI-MS analysis of CDC peptides in releasates

collected during an 8-CPT-cAMP-induced electrical dis-

charge of the CDC system in vitro directly showed that

depletion of CDC peptides from the COM is correlated to

their release. As the CDCs are activated directly in the in vitro

experiment whereas the clean water stimulus acts through

indirect pathways that eventually impinge on the CDCs, we

detected CDC peptides in the releasate as early as the first

15-min collection interval after 8-CPT-cAMP application. In

the second interval (15–30 min) the release rates of CTP and

d peptide were high and decreased thereafter, whereas the

release rates of CDCH remained high until 60 min. The

relatively early decreases in the release rates of the peptides

were accompanied by an increase of the truncated forms in the

releasate. The summation of the intact peptides and their

truncated forms, therefore, represents the true release rate of

the peptides and this may account for the apparent early

plateau of the release of these peptides. On the other hand, the

higher stability of CDCH underscores the hormonal function

of this peptide. In the last incubation interval of 60–90 min

the continuous release of CDC peptides could still be detected

after the discharge had ended. The appearance of CDC

peptides in the releasate during this latter interval probably

H F ( F H ) F Y G P Y D V F Q R D V a m i d e

5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 5 5 0 6 0 0

6 5 0 7 0 0 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 1 0 0 0 1 0 5 0 1 1 0 0 1 1 5 0 1 2 0 0

M/Z

Rel

ativ

eIn

tens

ity

1 2 5 0 1 3 0 0 1 3 5 0 1 4 0 0 1 4 5 0 1 5 0 0 1 5 5 0 1 6 0 0 1 6 5 0 1 7 0 0 1 7 5 0 1 8 0 0

7 0 .0

1 2 9 .1

1 6 6 .1

2 8 5 .2b2

4 3 2 .5

6 5 2 .4

9 1 2 .4b7

b5

b3

1 0 2 7 .5b8

1 1 2 6 .5b9

1 2 7 3 .0

b1 0

3 8 8 .0Y3

5 1 6 .0Y4

6 6 3 .0Y5

7 6 2 .7Y6

8 7 7 .0Y7 1 0 4 0 .0

Y8 1 1 3 5 .0Y9

1 1 9 4 .4Y1 0

1 3 5 7 .0Y1 1

1 5 0 3 .1Y1 2

1 7 8 9 .0

5 6 9Y9++

2 8 5 .24 3 2 .5

6 5 2 .4 1 0 2 7 .51 1 2 6 .5

1 2 7 3 .0

3 8 8 .05 1 6 .0

6 6 3 .07 6 2 .7

8 7 7 .01 0 4 0 .0

1 1 3 5 .01 3 5 7 .01 5 0 3 .1 1 1 9 4 .0

1 1 0 .1

1 2 0 .0

1 3 6 .18 8 .0

7 2

8 4

5 4 7 .0

(a)

(b)

Fig. 9 Structural characterization of the

molecule at m/z 1789 from the cerebral

commissure extract by high-energy colli-

sion-induced fragmentation tandem mass

spectrometry (MS). (a) MS/MS spectrum of

the molecular ion species at m/z 1789. (b)

Assignments of the fragment ions of the

peptide. Overlapping series of backbone

fragment ions of the b and y type are pre-

sent. For further explanations see text.



resulted from residual leakage of released CDC peptides from

the connective tissue surrounding the COM.

The truncated CDC peptides were not present in the CDCs

or COM, suggesting that they were cleaved after their

release. Enzymatic cleavage has an important role in the

termination of peptidergic signals (for review, see Nyberg

and Terenius 1990; Li 2001). For example, in the egg-laying

system of Aplysia, several proteolytic enzymes have been

implicated in the processing and inactivation of a-bag cell

peptide (Owens et al. 1992). Neutral metalloendopeptidase

cleaves a-bag cell peptide at Arg–Phe bonds and the

aminopeptidase subsequently removes Phe. These enzymes

may also be involved in cleavage of CTP at the Ala–Phe

bond (yielding CTP4)22) and subsequent removal of the next

available amino terminal Phe (yielding CTP5)22) (Fig. 8).

The amino terminal truncation, i.e. removal of Leu of CDCH,

may be the result of the activity of a Leu-aminopeptidase-like

enzyme (Fig. 8). As in Aplysia (Squire et al. 1991), this

enzyme may be present in the hemolymph of Lymnaea.

Our initial goal was to study the dynamics of the peptides

derived from the CDCH precursor. Interestingly, as the mass

spectrometry-based peptidomics analysis is an open screen-

ings approach, it allows for detection of novel peptides. It

now appears that the diversity in peptide expression in the

CDC system and the control of egg laying may be more

complex than hitherto suggested. For example, two novel

peptides of 1788 and 5895 Da exhibited changes in peptide

levels in vivo and in vitro, similar to the CDCH precursor

peptides. The sequence of the smaller peptide is novel and

was established as HF(FH)FYGPYDVFQRDVamide. Stud-

ies are in progress to elucidate the structures of the other

novel peptides contained in the CDC system and to

understand the role of these diverse peptides in the regulation

of the egg-laying processes.

Acknowledgements

The authors wish to thank Dr K.f. Medzirhadszky for interpretation

of the MS/MS spectrum and Ing. J.C. Lodder for fitting of the data

of Fig. 4 . In addition, Nederlandse Organisatie voor Wetenschap-

pelijk Onderzoek-Algemene Levenswetenschappen (NWO-ALW)

is acknowledged for providing financial support for the MALDI-

MS.

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Documents

Spatio-temporal dynamics of the egg-laying-inducing peptides during an egg-laying cycle: a semiquantitative matrix-assisted laser desorption/ionization mass spectrometry approach