Neuropeptidomic analysis of the brain and thoracic ganglion from the Jonah crab, Cancer borealis

Biochemical and Biophysical Research Communications 308 (2003) 535–544

www.elsevier.com/locate/ybbrc

BBRC

Neuropeptidomic analysis of the brain and thoracic ganglionfrom the Jonah crab, Cancer borealis

Jurgen Huybrechts,a,* Michael P. Nusbaum,b Luc Vanden Bosch,a Geert Baggerman,a

Arnold De Loof,a and Liliane Schoofsa

a Laboratory of Developmental Physiology and Molecular Biology, 59 Naamsestraat, BE-3000 K.U. Leuven, Belgiumb Department of Neuroscience, University of Pennsylvania, School of Medicine, Philadelphia, PA, USA

Received 2 July 2003

Abstract

Mass spectrometric methods were applied to determine the peptidome of the brain and thoracic ganglion of the Jonah crab

(Cancer borealis). Fractions obtained by high performance liquid chromatography were characterized using MALDI-TOF MS and

ESI-Q-TOF MS/MS. In total, 28 peptides were identified within the molecular mass range 750–3000 Da. Comparison of the mo-

lecular masses obtained with MALDI-TOF MS with the calculated molecular masses of known crustacean peptides revealed the

presence of at least nine allatostatins, three orcokinin precursor derived peptides, namely FDAFTTGFGHS, [Ala13]-orcokinin, and

[Val13]-orcokinin, and two kinins, a tachykinin-related peptide and four FMRFamide-related peptides. Eight other peptides were de

novo sequenced by collision induced dissociation on the Q-TOF system and yielded AYNRSFLRFamide, PELDHVFLRFamide or

EPLDHVFLRFamide, APQRNFLRFamide, LNPFLRFamide, DVRTPALRLRFamide, and LRNLRFamide, which belong to

the FMRFamide related peptide family, as well as NFDEIDRSGFA and NFDEIDRSSFGFV, which display high sequence

similarity to peptide sequences within the orcokinin precursor of Orconectes limosus. Our paper is the first (neuro)peptidomic

analysis of the crustacean nervous system.

� 2003 Elsevier Inc. All rights reserved.

Keywords: Q-TOF; MALDI-TOF imaging; Mass spectrometry; Peptidomics; Cancer borealis; Orcokinin; FMRFamide-related peptides;

Allatostatins

Neuropeptides are important messenger molecules

that selectively influence most, if not all physiological

processes including behavior. They act as neurotrans-

mitters, modulators, and classical hormones. When re-

leased into the circulation they often display pleiotropic

functions. Our knowledge of the structure and function

of arthropod neuropeptides has been gradually in-

creasing over the past three decades. A dramatic in-crease was seen shortly after the completion and

publication of the Drosophila genome project in 2000 [1].

To illustrate this revolution: in 1975 not less than

125,000 cockroaches were sacrificed to purify and

characterize a single neuropeptide (proctolin) [2]. In

contrast, very recently (mid 2002), biochemical evidence

* Corresponding author. Fax: +32-16-32-39-02.

E-mail address: [email protected] (J. Hu-

ybrechts).

0006-291X/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/S0006-291X(03)01426-8

was provided for the presence of 28 neuropeptides in the

larval Drosophila central nervous system, using a com-

bination of MS and bioinformatics [3]. In this recent

study, only 50 CNSs of the fruit fly were needed to

obtain enough material to be able to characterize these

peptides.

The genome project is not the only driving force of

neuropeptide research. In the 1980s Electrospray Ioni-zation [4] and Matrix Assisted Laser Desorption/Ioni-

zation [5] were introduced in mass spectrometry

applications. These so called “soft” ionization tech-

niques made the volatilization of biomolecules possible

without dissociating them. By applying these techniques

to peptide research, peptides can be isolated and iden-

tified from smaller tissue extracts [6]. Even entire peptide

profiles from specified organs can be studied [3,7,8].Tissue extraction is not always necessary because small

organs [9], single cells [10] or even individual cell

mail to: [email protected]

Fig. 1. Fractionation of an extract, containing 50 brain–thoracic

ganglion equivalents of Cancer borealis. Chromatographic conditions:

an XTerra C18 column (7.8 mm� 300mm; particle size 10lm) was

eluted with a linear gradient of 0.1% TFA (solvent A) and 0.1% TFA/

60% acetonitrile (solvent B). The gradient ran from 0% A to 100% B

within 60min at a flow rate of 1 ml/min. The eluting peptides were

monitored with a variable wavelength UV detector, set at 214 nm.

Fractions were collected every minute, their numbers corresponding to

the elution time (fraction 1 eluting between 0 and 1min).

536 J. Huybrechts et al. / Biochemical and Biophysical Research Communications 308 (2003) 535–544

organelles [11] can be studied directly with MALDI-TOF MS.

To date several crustacean neuropeptide families have

been described. Some of these families show significant

similarities with peptide families of other well-studied

arthropods: the class of insects.

The nonapeptide crustacean cardioactive peptide

(CCAP) was first isolated and characterized from the

pericardial organs of the shore crab Carcinus maenas

[12] and shown to be widely distributed in both crusta-

ceans and insects [13]. CCAP has a potent myostimu-

lating effect on the crustacean hindgut [14] and has been

associated with several physiological functions in both

insects and crustaceans such as cardiac control, visual

modulation, locomotion, and ecdysis.

Proctolin was isolated from the perisympathic organs

of Homarus americanus [15] and C. maenas [16]. Similarto CCAP, proctolin shows a myostimulating effect on

the crayfish hindgut [17].

The chromatophorotropins, red pigment concentrat-

ing hormone (RPCH), and pigment dispersing hormone

(PDH) were isolated from the X-organ—sinus gland

system. RPCH is an octapeptide originally sequenced

from Pandalus borealis [18]. Its structure is fully pre-

served in a wide range of crustacean species [19] andbears great resemblance to that of the later elucidated

adipokinetic hormones of insects. In 1976, Fernlund

isolated and sequenced an octodecapeptide, which had

the opposite effect on pigments in the epidermal chro-

matophors and therefore was called PDH [20].

The thoracic ganglia of crustaceans proved to be a

rich source for neuropeptides belonging to the allatost-

atin superfamily [21–23]. All these crustacean allatosta-tins have an inhibitory effect on the crustacean hindgut

contractility assay. Allatostatins are present in several

invertebrate classes (amongst for example insects and

plathyhelminths). Their myomodulatory function ap-

pears to be conserved through evolutionary time. The

juvenile hormone inhibitory activity, another physio-

logical function, appears to be confined to specific insect

orders [24].Several members from the FMRFamide superfamily

have been identified in decapods [25–30]. All the pep-

tides proved to be myotropic/cardioactive and were

isolated from the eyestalks or the pericardial organs.

Also in insects, the FMRFamide-related peptides form a

large and multifunctional family of structurally related

neuropeptides [31].

The kinin peptide family is also widely distributed ininvertebrates [32]. They were initially isolated from in-

sects, later from the mollusk, Lymnaea stagnalis, and

most recently from the crustacean, Penaeus vannamei.

Tachykinins (related to vertebrate substance P) share a

conserved C-terminal sequence, they have been identi-

fied in several invertebrate species including crustaceans

[33].

One peptide family is limited to crustaceans and hasnot (yet) been demonstrated in insects thus far: the

orcokinins and orcomytropin, which is derived from the

same precursor [34–36]. They were isolated from an

abdominal nerve cord extract by their effect on the vis-

ceral muscles of the crustacean hindgut.

A complete investigation of the entire neuropepti-

dome (all expressed neuropeptides) of a crustacean has,

in contrast to insects [3,7], not yet been conducted.Therefore, we analyze the neuropeptidome of the brain

and thoracic ganglia of the Jonah crab, Cancer borealis.

HPLC fractions were screened for the presence of

putative neuropeptides applying MALDI-TOF and

ESI-Qqoa-TOF mass spectrometric methods. The

MALDI-TOF is more sensitive, whereas the ESI-Qqoa-

TOF allowed obtaining sequence information using

collision induced dissociation (CID). This technique isvery useful for peptides having a maximum molecular

mass below 3000 Da and yields fragmentation spectra

that are relatively easy to interpret.

Materials and methods

Tissue extraction and Sep-Pak purification. The brains and thoracic

ganglia of 50 C. borealis crabs were removed and homogenized in a

solution of methanol:water:acetic acid (90:9:1) immediately upon dis-

section. The tissue was then sonicated and subsequently centrifuged at

9000g for 10min. More extraction medium was added to the pellet and

this was again sonicated and centrifuged at 9000g for 10min. Both

supernatants were pooled and stored at 4 �C. After evaporation of the

extraction solvent, the samples were delipidated using ethylacetate and

n-hexane [37]. Procedures for solid phase extraction have been previ-

ously described [37]. All aqueous solutions used for solid phase ex-

tractions and HPLC analysis contained 0.1% trifluoroacetic acid

J. Huybrechts et al. / Biochemical and Biophysical Research Communications 308 (2003) 535–544 537

(TFA), as ion paring agent. The dry residue of the extracted material

of 50 brain and thoracic ganglion equivalents was dissolved in 5 ml of

0.1% TFA and applied to Sep-Pak cartridge. This cartridge was eluted

with 5 ml of 60% acetonitrile/water and the resulting fraction was

lyophilized.

High performance liquid chromatography. HPLC analysis was per-

formed on a Gilson liquid chromatograph equipped with two pumps

and a solvent mixing unit. The eluting peptides were detected with a

variable wavelength UV detector, set at 214 nm (Waters 486 Tunable

Absorbance detector). The separation of the compounds was carried

out on a semi-preparative Waters XTerra C18 column (7.8mm�300mm; particle size 10lm) at ambient temperature and with a sol-

vent flow rate of 2ml/min. The lyophilized residue from 50 brain and

thoracic ganglion equivalents was dissolved in 1.5ml of 0.1% TFA and

transferred to the injector after filtering through a Millipore 0.22 lm

filter. Immediately after injection a linear gradient from 0% to 60%

acetonitrile containing 0.1% TFA was initiated. Final concentration

(60% acetonitrile) was achieved after 60min. Fractions containing 2ml

were collected every minute starting at 0 until 60min.

MALDI-TOF mass spectrometry. Matrix-assisted laser desorption/

ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS)

was performed on a Reflex IV (Bruker Daltonic GmbH, Germany),

Fig. 2. MALDI-TOF mass spectra from fractions 25, 32, 33, and 38, monoiso

novo sequenced using ESI-Qqoa-TOF MS, ion peaks indicated with * are fr

equipped with a N2 laser and pulsed ion extraction accessory. From

every fraction 100ll was taken, lyophilized, and resuspended in a 5ll

solution of water:acetonitrile:formic acid (50:49:0.1). One ll of this

solution (representing 0.5 equivalent, the remaining 4ll was stored at

)20 �C) was transferred to a ground steel target plate and mixed with

0.5 ll of a saturated solution of a-cyano-4-hydroxycinnamic acid in

ethanol:acetonitrile (50:50), and dried under a gentle flow of argon to

speed up the evaporation of solvents to minimize crystal sizes. The

instrument was calibrated using a standard peptide mixture, contain-

ing angiotensin II (1045.54Da), angiotensin I (1295.68Da), substance

P (1346.73Da), bombesin (1618.82Da), ACTH clip 1–17 (2092.08Da),

and ACTH clip 18–39 (2464.19Da), (Bruker Daltonic GmbH,

Germany). Spectra were recorded in the reflectron mode within a

mass range from m/z 500 to m/z 3000 and are the results of 20–100

shots.

Further investigation of fractions by means of Q-TOF mass spec-

trometry. Nanoflow electrospray ionization (ESI) double quadrupole

(Qq) orthogonal accelaration (oa) time-of-flight (TOF) mass spec-

trometry (MS) was performed on a Q-TOF system (Micromass, UK).

Two microliter (representing 1 equivalent, taken from the sample so-

lution prepared previously for MALDI-TOF MS) was loaded into a

gold-coated capillary (Long NanoES spray capillaries for Micromass

topic [M+H]þ are given in the spectra. Underlined sequences were de

agments of cryptocyanin.

Table 1

Peptides identified in a brain–thoracic ganglion extract from Cancer borealis by mass spectrometric techniques

Peptide name Peptide sequence Molecular mass (Monoisotopic) Fraction

Measured Theoretical

Allatostatins

Carcinustatin 2 EAYAFGLamide 768.3 768.4 41

Carcinustatin 5 NPYAFGLamide 779.3 779.4 22/25

Carcinustatin 8 AGPYAFGLamide 793.4 793.4 27

Carcinustatin 9 GGPYAFGLamide 779.3 779.4 22/25

Carcinustatin 19 APTDMYSFGLamide 1099.5 1099.5 24

Penaeustatin 12 SDYAFGLamide 770.4 770.4 43

Penaeustatin 19 GHYNFGLamide 805.4 805.4 17

Penaeustatin 22 AAPYEFGLamide 865.3 865.4 25

Penaeustatin 34 pQNMYSFGLamide 940.3 940.4 25

RFamides

NF 1 NRNFLRFamide 964.6 964.5 31/32

Cab-FLP 1 TNRNFLRFamide 1065.6 1065.6 31

Pem-FLP 5 SMPSLRLRFamide 1104.5 1104.6 34

[Val2]-FLP 6 DVRTPALRLRFamide 1341.9 1341.8 32

Pem-FLP 7 GYRKPPFNGSIFamide 1380.7 1380.7 33

Pem-PYF 3 YAIAGRPRFamide 1048.7 1048.6 18

[Ala1]-FaRP AYNRSFLRFamide 1171.9 1171.6 33

[Glu2–Leu3]-SchistoFLRFa PELDHVFLRFamide 1270.8 1270.7 39

APQRNFLRFamide 1146.8 1146.6 32

LNPFLRFamide 904.6 904.5 28

LRNLRFamide 816.6 816.5 30

Kinins

Pev-Kinin 2 DFSAWAamide 694.4 694.3 48

Pev-Kinin 4 VAFSPWGamide 761.3 761.4 16

Tachykinin related peptide APSGFLGMRamide 933.50 933.49 31

Orcokinin like peptides

FDAFTTGFGHS 1185.5 1185.5 33

NFDEIDRSGFA 1269.8 1269.6 33

[Ala13]-orcokinin NFDEIDRSGFGFA 1473.7 1473.7 36

[Val13]-orcokinin NFDEIDRSGFGFV 1501.8 1501.7 38

[Ser9–Val13]-orcokinin FDEIDRSSFGFV 1531.8 1531.7 38

Peptide sequences in bold were sequenced using ESI-Qqoa-TOF MS/MS, underlined sequences were sequenced de novo. All measured molecular

masses that were present in MALDI-TOF spectra are in italics.


Q-TOF, Proxeon Biosystems A/S, Denmark). The sample was sprayed

at a typical flow rate of 30 nl/min giving extended analysis time in

which MS spectra as well as several MS2 spectra were acquired. During

MS2 or tandem mass spectrometry, fragment ions are generated from a

selected precursor ion by collision-induced dissociation (CID). Since

not all peptide ions fragment with the same efficiency the collision

energy is typically varied between 20 and 35V so that the parent ion is

fragmented into a satisfying number of different daughter ions. The

amino acid sequence is determined by calculating the m/z difference

(which corresponds to the mass of an amino acid residue) between the

adjacent y-ion peaks and/or b-ion peaks.

Results and discussion

HPLC separation on the XTerra C18 column of an

extract of 50 brain–thoracic ganglion equivalents, ini-

tially prepurified on a C18 reverse phase Sep-pak, yielded

a satisfying separation of components, bearing in mindthat no pure fractions were necessary for further anal-

ysis (Fig. 1). All fractions were screened for the presence

of neuropeptides using MALDI-TOF-MS. Examples of

spectra of individual fractions are shown in Fig. 2.

Allatostatins

So far 58 different naturally occurring isoforms of

allatostatins are identified in crustaceans. Twenty alla-

tostatins were isolated from the shore crab, C. maenas

[21], 39 in the tiger prawn, Penaeus monodon [22], andthree in the crayfish Orconectes limosus [24]. Based on

mass spectral data we conclude that in C. borealis at

least nine putative isoforms of allatostatins are present,

five of them being identical to those previously identified

in C. maenas and four identical to those previously

identified in P. monodon. These peptides occurred as low

intensity ion peaks in the MALDI-TOF spectra and

were unfortunately not seen in ESI-Qqoa-TOF spectra,nor did any of the ions selected for fragmentation lead

to the identification of new allatostatin isoforms. Other

peptides identified based solely on the appearance of

Fig. 3. Collision induced dissociation spectra of two de novo sequenced members of the RFamide neuropeptide family. The upper panel showing the

fragmentation of LNPFLRFamide and the lower panel showing the fragmentation of LRNLRFamide. a-type, b-type, y-type, and z-type fragment

ions are indicated in the tables. The theoretical fragment ion masses found in the spectrum are indicated in bold. Mass differences between expected

and observed fragment ions are indicated by d.


their corresponding ions in the MALDI TOF survey

were Pev-Kinin 2, Pev-Kinin 4, and the tachykinin

related peptide (Table 1).

FMRFamide-related peptides

One of the most phylogenetically diverse, but struc-

turally similar, family of peptides is the family that

contains the FMRFamide-related peptides. The tetra-peptide, FMRFamide, was the first member to be se-

quenced and was identified as a cardioacceleratory

peptide in the clam, Macrocalista nimbosa [38].

FMRFamide is now regarded as the primary member of

an extensive family of peptides with diverse biological

functions. In crustaceans FMRFamide-like peptides

have been isolated and characterized from pericardial

organs, thoracic ganglia, and from eyestalks [25–30].

TNRNFLRFamide and SDRNFLRFamide were iso-

lated and sequenced from the stomatogastric nervous

system of the crab C. borealis [39]. All these peptides

display versatile physiological functions in crustaceans[40]. Here we provide biochemical evidence for the

presence of at least 11 FMRFamide-related peptides in a

brain–thoracic ganglion extract of C. borealis. NR

NFLRFamide (F32, m/z: 965.6, Fig. 2), TNRNFLR

Famide (F31, m/z: 1066.6), SMPSLRLRFamide, GYR

KPPFNGSIFamide (F33, m/z: 1381.7), and YA-

IAGRPRFamide (F18, m/z: 1049.7) have been charac-

terized previously in other crustacean species (onlyNRNFLRFamide was identified in C. borealis). Most of

these peptides occurred also as double charged ions in

the Q-TOF spectra and were selected for fragmentation.

The resulting fragmention spectra (raw data treated with

MaxEnt3 Software, Micromass, UK) confirmed the


fragmentation of AYNRSFLRFamide and the lower panel showing the fragmentation of DVRTPALRLRFamide. a-type, b-type, y-type, and z-type

fragment ions are indicated in the tables. The theoretical fragment ion masses found in the spectrum are indicated in bold. Mass differences between

expected and observed fragment ions are indicated by d.


results obtained by MALDI-TOF MS (Table 1). In

addition, six members of the FMRFamide-related pep-

tide family were sequenced de novo using this approach.

These sequences were assigned as LNPFLRFamide

(Fig. 3 upper panel), LRNLRFamide (Fig. 3 lower pa-

nel), AYNRSFLRFamide (Fig. 4 upper panel), DVRTP

ALRLRFamide (Fig. 4 lower panel), PELDHVFLR

Famide or EPLDHVFLRFamide (Fig. 5 upper panel),and APQRNFLRFamide (Fig. 5 lower panel). AYN

RSFLRFamide and DVRTPALRLRFamide are, ex-

cept for one amino acid, identical to the previously

identified peptides GYNRSFLRFamide (Callinectes

sapidus) and DGRTPALRLRFamide (P. monodon),

respectively. For one peptide the MS/MS spectrum

could be read as EPL DHVFLRFamide or PELDHV

FLRFamide, the feature that a proline residue in posi-

tion 2 protects the N-terminus from general aminopep-

tidases [41] favors the first sequence. Anyway, the C-

terminal (-DHVFLRFamide) is completely identical to

the C-terminal of myosuppressins of other, non-crusta-

cean arthropods including cockroaches, flies, and

grasshoppers [42].

Orcokinins

Orcokinin, NFDEIDRSGFGFN, was the first iden-

tified member (isolated from 1200 abdominal nerve

cords of the crayfish O. limosus) of a peptide family

specific for crustaceans [34]. Activity studies showed thatorcokinins are potent hindgut-stimulating factors [43].

Immunocytochemical studies showed the presence of

orcokinin in all parts of the nervous system [44]. To


fragmentation of PE(EP)LDHVFLRFamide and the lower panel showing the fragmentation of APQRNFLRFamide. a-type, b-type, y-type, and z-

type fragment ions are indicated in the tables. The theoretical fragment ion masses found in the spectrum are indicated in bold. Mass differences

between expected and observed fragment ions are indicated by d.


date, in total six different orcokinin isoforms are iden-

tified in several crustacean species namely orcokinin,

[Ser9]-orcokinin, [Ala13]-orcokinin, [Val13]-orcokinin

[43], [Thr8–His13]-orcokinin, FDAFTTGFGHS [45],

and [Ala8–Ala13]-orcokinin [46]. Each crustacean species

contains more than one orcokinin isoform. Variations in

isoforms (inter and intra species) are related to point

mutations at the DNA level. In C. borealis we foundbiochemical evidence for the presence of orcokinin (F33,

m/z: 1186.5, Fig. 2), [Ala13]-orcokinin (F36, m/z: 1473.7),

and [Val13]-orcokinin (F38, m/z: 1502.8, Fig. 2). Two

additional orcokinin isoforms, [Ser9–Val13]-orcokinin

(m/z: 766.91, Fig. 6 lower panel) and NFDEIDRSGFA

(m/z: 635.90, Fig. 6 upper panel), were sequenced de

novo. The latter is a shorter peptide than the “classical”

orcokinins, with the amino acids at position 11 and 12

from the original peptide apparantly deleted. These re-

sults are summarized in Table 1.

Protein fragments

Besides peptides, a number of fragments were se-

quenced as well (KIFEPLR, YKIFEPL, YKIFEPLR,

YKIFEPLRS, YKIFEPLRSN, and YKIFEPLRSNL).

These fragments were all present as high intenstity peaks

in the MALDI-TOF spectra (Fig. 2). NCBI BLAST

search revealed that they are fragments of cryptocyanin.

Cryptocyanin was isolated and characterized in Cancer

magister from hepatopancreas tissue [47]. The concen-tration of cryptocyanin in the hemolymph is closely

coordinated with the molt cycle and peaks in late pre-

molt. It is not clear why these fragments appear in a

Fig. 6. Collision induced dissociation spectra of two de novo sequenced orcokinin like neuropeptides. The upper panel showing the fragmentation of

NFDEIDRSGFA and the lower panel showing the fragmentation of NFDEIDRSSFGFV. a-type, b-type, y-type, and z-type fragment ions are

indicated in the tables. The theoretical fragment ion masses found in the spectrum are indicated in bold. Mass differences between expected and

observed fragment ions are indicated by d.


brain–thoracic ganglion extract. This could be due to

contamination of the brain–thoracic ganglion extract

with hemolymph.

Several crustacean neuropeptides within the mass

range of 750–3000 Da were not seen in our peptidomicapproach (ion peaks corresponding to proctolin and

the enkephalins, below 750 Da, were also actively

searched for but not found). This could be attributed

to the following factors: they are not expressed in the

brain–thoracic ganglion (RPCH and PDH), their con-

centration may be below the sensitivity of the used

instrumental setup (CCAP), and not all peptides are

extracted or ionize with the same efficiency. Severalpeptide ion peaks of low intensity that were fragmented

could not be sequenced because of the low quality and

signal intensity of the obtained daughter ions. Never-

theless, the combined use of HPLC fractionation and

mass spectrometric analysis by MALDI-TOF MS and

Q-TOF MS proves to be an elegant tool to analyse the

peptidome of the crustacean nervous system. Starting

from a relative small amount of tissue we were able to

identify 28 peptides, members of four different peptidefamilies, of which eight were sequenced de novo. The

ability to survey an animal for putative neuropeptides

is important for improving our knowledge on the ex-

pression of these signalling molecules within an or-

ganism. A disadvantage, inherent to the mass

spectrometric methods used, is the inability to distin-

guish unequivocally between certain amino acids such

as Gln/Lys and Ile/leu. Therefore, the given amino acidsequences in this study were based on obtained se-

quence data combined with sequences resulting from

NCBI BLAST searches, minimizing errors against Gln/

Lys and Ile/Leu.


Acknowledgments

We thank the members from the laboratory of Michael P. Nus-

baum (Dept. of Neuroscience, University of Pennsylvania school of

Medicine) for providing the C. borealis tissue used in this experiment.

This project was sponsored by the Flemish Science Foundation (Fonds

voor Wetenschappelijk Onderzoek-Vlaanderen, FWO, G.0187.00 and

G.0175.02), and a grant from the National Insitute of Neurological

Disorders and Stroke (NIH NS29436) to M.P.N. J.H. benefits from a

scholarship from the FWO.

References

[1] M.D. Adams et al., The genome sequence of Drosophila melano-

gaster, Science 287 (2000) 2185–2195.

[2] A.N. Starratt, B.E. Brown, Structure of the pentapeptide proct-

olin, a proposed neurotransmitter in insects, Life Sci. 17 (1975)

1253–1256.

[3] G. Baggerman, A. Cerstiaens, A. De Loof, L. Schoofs, Peptido-

mics of the larval Drosophila melanogaster central nervous system,

J. Biol. Chem. 277 (2002) 40368–40374.

[4] M. Yamashita, J.B. Fenn, Negative ion production with the

electrospray ion source, J. Phys. Chem. 88 (1984) 4671–4675.

[5] M. Karas, F. Hillenkamp, Laser desorption ionisation of proteins

with molecular masses exceeding 10 000 Daltons, Anal. Chem. 60

(1988) 1299–1301.

[6] D. Veelaert, G. Baggerman, R. Derua, E. Waelkens, T. Meeusen,

G. Van de Water, A. De Loof, L. Schoofs, Identification of a new

tachykinin from the midgut of the desert locust, Schistocerca

gregaria, by ESI-Qq-oa-TOF mass spectrometry, Biochem. Bio-

phys. Res. Commun. 266 (1999) 237–242.

[7] E. Clynen, G. Baggerman, D. Veelaert, A. Cerstiaens, D. Van der

Horst, L. Harthoorn, R. Derua, E. Waelkens, A. De Loof, L.

Schoofs, Peptidomics of the pars intercerebralis–corpus cardiacum

complex of the migratory locust, Locusta migratoria, Eur.

J. Biochem. 268 (2001) 1929–1939.

[8] E. Clynen, J. Huybrechts, A. De Loof, L. Schoofs, Mass

spectrometric analysis of the perisympathetic organs in locusts:

identification of novel periviscerokinins, Biochem. Biophys. Res.

Commun. 300 (2003) 422–428.

[9] R. Predel, Peptidergic neurohemal system of an insect: mass

spectrometric morphology, J. Comp. Neurol. 436 (2001) 363–

375.

[10] L. Li, R.W. Garden, E.V. Romanova, J.V. Sweedler, In situ

sequencing of peptides from biological tissues and single cells

using MALDI-PSD/CID analysis, Anal. Chem. 71 (1999) 5451–

5458.

[11] S.S. Rubakhin, R.W. Garden, R.R. Fuller, J.V. Sweedler,

Measuring the peptides in individual organelles with mass

spectrometry, Nat. Biotechnol. 18 (2000) 172–175.

[12] J. Stangier, C. Hilbich, K. Beyreuther, R. Keller, Unusual

cardioactive peptide (CCAP) from pericardial organs of the shore

crab, Carcinus maenas, Proc. Natl. Acad. Sci. USA 84 (1987) 575–

580.

[13] H. Dircksen, Distribution and physiology of the crustacean

cardioactive peptide in arthropods, in: K.G. Davey, R.E. Peter,

S.S. Tobe (Eds.), Perspectives in Comparative Endocrinology,

National Research Council of Canada, Ottawa, 1994, pp. 138–

148.

[14] H. Dircksen, R. Keller, Immunocytochemical localization of

CCAP, a novel crustacean cardioactive peptide, in the nervous

system of the shore crab, Carcinus maenas L., Cell Tissue Res. 254

(1988) 347–360.

[15] T.L. Schwarz, G.M.H. Lee, K.K. Siwicki, D.G. Standaert, E.A.

Kravitz, Proctolin in the lobster: the distribution, release and

chemical characterization of a likely neurohormone, J. Neurosci. 4

(1984) 1300–1311.

[16] J. Stangier, H. Dircksen, R. Keller, Identification and immu-

nocytochemical localization of proctolin in pericardial organs

of the shore crab, Carcinus maenas, Peptides 7 (1986) 67–72.

[17] A.J. Mercier, A.B. Lange, V. TeBrugge, I. Orchard, Evidence for

proctolin-like and RFamide-like neuropeptides associated with

the hindgut of the crayfish Procambarus clarkii, Can. J. Zool. 75

(1997) 1208–1225.

[18] P. Fernlund, L. Josefsson, Crustacean color change hormone:

amino acid sequence and chemical synthesis, Science 177 (1972)

173–175.

[19] G. Gaus, L.H. Kleinholz, G. Kegel, R. Keller, Isolation and

characterization of red-pigment-concentrating hormone (RPCH)

from six crustacean species, J. Comp. Physiol. B 160 (1990) 373–

379.

[20] P. Fernlund, Structure of a light-adapting hormone from the

shrimp Pandalus borealis, Biochim. Biophys. Acta 439 (1976) 17–

25.

[21] H. Duve, A.H. Johnsen, J.L. Maestro, A.G. Scott, P.P. Jaros, A.

Thorpe, Isolation and indentification of multiple neuropeptides of

the allatostatin superfamily in the shore crab Carcinus maenas,

Eur. J. Biochem. 250 (1997) 727–734.

[22] H. Duve, A.H. Johnsen, A.G. Scott, A. Thorpe, Allatostatins of

the tiger prawn, Penaeus monodon (Crustacea: Penaeidea), Pep-

tides 23 (2002) 1039–1051.

[23] H. Dircksen, P. Skiebe, B. Abel, H. Agricola, K. Buchner, J.E.

Muren, D.R. N€aassel, Structure, distribution, and biological

activity of novel members of the allatostatin family in the crayfish

Orconectes limosus, Peptides 20 (1999) 695–712.

[24] W.G. Bendena, B.C. Donly, S.S. Tobe, Allatostatins: a growing

family of neuropeptides with structural and functional diversity,

Ann. N. Y. Acad. Sci. 897 (1999) 311–329.

[25] B.A. Trimmer, L.A. Kobierski, E.A. Kravitz, Purification and

characterization of FMRFamide-like immunoreactive substances

from the lobster nervous system: isolation and sequence analysis

of two closely related peptides, J. Comp. Neurol. 266 (1987) 16–

26.

[26] K.G. Krajniak, The identification and stucture-activity relations

of a cardioactive FMRFamide-related peptide from the blue crab

Callinectes sapidus, Peptides 12 (1991) 1295–1302.

[27] A.J. Mercier, I. Orchard, V. TeBrugge, M. Skerret, Isolation of

two FMRFamide-related peptides from crayfish pericardial

organs, Peptides 14 (1993) 137–143.

[28] P. Sithigorngul, W. Saraithonkum, S. Jaideechoey, S. Longy-

ant, W. Sithigorngul, Novel FMRFamide-like neuropeptides

from the eyestalk of the giant freshwater prawn Macrobrach-

ium rosenbergii, Comp. Biochem. Physiol. B 120 (1998) 587–

595.

[29] P. Sithigorngul, W. Saraithonkum, S. Longyant, N. Panchan, W.

Sithigorngul, A. Petsom, Three more FMRF-amide-like neuro-

peptide sequences from the giant freshwater prawn Macrobrach-

ium rosenbergii, Peptides 22 (2001) 191–197.

[30] P. Sithigorngul, J. Pupuem, C. Krungkasem, S. Longyant, P.

Chaivisuthangkura, W. Sithigorngul, A. Petsom, Seven novel

FMRFamide-like neuropeptide sequences from the eyestalk of the

giant tiger prawn Penaeus monodon, Comp. Biochem. Physiol. B

131 (2002) 325–337.

[31] I. Orchard, A.B. Lange, W.G. Bendena, FMRFamide-related

peptides: a multifunctional family of structurally related neuro-

peptides in insects, Adv. Insect Physiol. 28 (2001) 267–329.

[32] P. Torfs, J. Nieto, D. Veelaert, D. Boon, G. Van de Water, E.

Waelkens, R. Derua, J. Calderon, A. De Loof, L. Schoofs, The

kinin peptide family in invertebrates, Ann. N. Y. Acad. Sci. 897

(1999) 361–373.

[33] J. Nieto, D. Veelaert, R. Derua, E. Waelkens, A. Cerstiaens, G.

Coast, B. Devreese, J. Van Beeumen, J. Calderon, A. De Loof, L.


Schoofs, Identification of one tachykinin- and two kinin-related

peptides in the brain of the white shrimp, Penaeus vannamei,

Biochem. Biophys. Res. Commun. 248 (1998) 406–411.

[34] J. Stangier, C. Hilbich, S. Burdzik, R. Keller, Orcokinin: a novel

myotropic peptide from the nervous system of the crayfish,

Orconectes limosus, Peptides 13 (1992) 859–864.

[35] D. Bungart, C. Hilbich, H. Dircksen, R. Keller, Occurrence of

analogs of the myotropic neuropeptide orcokinin in the shore crab

Carcinus maenas: evidence for a novel neuropeptide family,

Peptides 16 (1995) 67–72.

[36] H. Dircksen, S. Burdzik, A. Sauter, R. Keller, Two orcokinins and

the novel octapeptide orcomyotropin in the hindgut of the crayfish

Orconectes limosus: identified myostimulatory neuropeptides orig-

inating together in neurones of the terminal abdominal ganglion,

J. Exp. Biol. 203 (2000) 2807–2818.

[37] L. Schoofs, G.M. Holman, T.K. Hayes, A. Tips, R.J. Nachman,

F. Vandesande, A. De Loof, Isolation, identification and synthesis

of locustamyotropin (Lom-MT), a novel biologically active insect

peptide, Peptides 11 (1989) 427–433.

[38] D.A. Price, M.J. Greenberg, Structure of a molluscan cardioex-

citatory neuropeptide, Science 197 (1977) 670–671.

[39] J.M. Weimann, E. Marder, B. Evans, R.L. Calabrese, The effects

of SDRNFLRFamide and TNRNFLRFamide on the motor

patterns of the stomatogastric ganglion of the crab Cancer

borealis, J. Exp. Biol. 181 (1993) 1–26.

[40] A.J. Mercier, R. Friedrich, M. Boldt, Physiological functions of

FMRFamide-like peptides (FLPs) in crustaceans, Microsc. Res.

Tech. 60 (2003) 313–324.

[41] R.E. Isaac, R.J. Siviter, P. Stancombe, D. Coates, D. Shirras,

Conserved roles for peptidases in the processing of invertebrate

neuropeptides, Biochem. Soc. Trans. 28 (2000) 460–464.

[42] L. Schoofs, G.M. Holman, L. Paemen, D. Veelaert, M. Ame-

linckx, A. De Loof, Isolation, identification, and synthesis of

PDVDHFLRFamide (SchistoFLRFamide) in Locusta migratoria

and its association with the male accessory glands, the salivary

glands, the heart, and the oviduct, Peptides 14 (1993) 409–421.

[43] D. Bungart, G. Kegel, S. Burdzik, R. Keller, Structure–activity

relationships of the crustacean myotropic neuropeptide orcokin,

Peptides 16 (1994) 199–204.

[44] D. Bungart, H. Dircksen, R. Keller, Quantitative-determination

and distribution of the myotropic neuropeptide orcokinin in the

nervous-system of astacidean crustaceans, Peptides 15 (1994) 393–

400.

[45] Y. Yasuda-Kamatani, A. Yasuda, Identification of orcokinin

gene-related peptides in the brain of the crayfish Procambarus

clarkii by the combination of MALDI-TOF and on line capillary

HPLC/Q-TOF mass spectrometries and molecular cloning, Gen.

Comp. Endocrinol. 118 (2000) 161–172.

[46] P. Skiebe, M. Dreger, M. Meseke, J.F. Evers, F. Hucho,

Identification of orcokinins in single neurons in the stomatogastric

nervous system of the crayfish, Cherax destructor, J. Comp.

Neurol. 444 (2002) 245–259.

[47] N.B. Terwilliger, L. Dangott, M. Ryan, Cryptocyanin, a crusta-

cean molting protein: evolutionary link with arthropod hemocy-

anins and insect hexamerins, Proc. Natl. Acad. Sci. USA 96 (1999)

2013–2018.

Neuropeptidomic analysis of the brain and thoracic ganglion from the Jonah crab, Cancer borealis

Documents