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omparative peptidomics of Caenorhabditis elegans versus C.riggsae by LC–MALDI-TOF MS

teven J. Husson a,*, Bart Landuyt a, Thomas Nys a, Geert Baggerman b, Kurt Boonen a,lke Clynen a, Marleen Lindemans a, Tom Janssen a, Liliane Schoofs a

Functional Genomics and Proteomics Unit, Department of Biology, K.U.Leuven, Naamsestraat 59, B-3000 Leuven, Belgium

ProMeta, Interfacultary Center for Proteomics and Metabolomics, K.U.Leuven O&N 2, Herestraat 49, B-3000 Leuven, Belgium

r t i c l e i n f o

rticle history:

eceived 29 May 2008

eceived in revised form

0 July 2008

ccepted 30 July 2008

ublished on line 7 August 2008

eywords:

europeptide

eptidomics

ass spectrometry

ALDI-TOF MS

p

lp

a b s t r a c t

Neuropeptides are important signaling molecules that function in cell–cell communication

as neurotransmitters or hormones to orchestrate a wide variety of physiological conditions

and behaviors. These endogenous peptides can be monitored by high throughput pepti-

domics technologies from virtually any tissue or organism. The neuropeptide complement

of the soil nematode Caenorhabditis elegans has been characterized by on-line two-dimen-

sional liquid chromatography and quadrupole time-of-flight tandem mass spectrometry

(2D-nanoLC Q-TOF MS/MS). Here, we use an alternative peptidomics approach combining

liquid chromatography (LC) with matrix-assisted laser desorption ionization time-of-flight

(MALDI-TOF) mass spectrometry to map the peptide content of C. elegans and another

Caenorhabditis species, Caenorhabditis briggsae. This study allows a better annotation of

neuropeptide-encoding genes from the C. briggsae genome and provides a promising basis

for further evolutionary comparisons.

# 2008 Elsevier Inc. All rights reserved.

avai lab le at www.sc iencedi rec t .com

journal homepage: www.elsev ier .com/ locate /pept ides

n

1. Introduction

The transparent, free-living, non-parasitic soil roundworm

Caenorhabditis elegans can be considered as the ideal model

system due to its short life cycle and its extremely well-defined

anatomy containing exactly 959 cells that are ordered in fully

differentiated tissues. It takes about 3 days from egg to egg and

the nematode goes through four larval stages (L1–L4) before

reaching adulthood. Hermaphrodites can self-fertilize or mate

with males in order to produce over 300 offspring. C. elegans

was the first multi cellular organism to have its genome fully

sequenced, about 10 years ago [47]. Its genome (about 100 Mb)

* Corresponding author. Tel.: +32 16 323904; fax: +32 16 323902.E-mail address: Steven.husson@bio.kuleuven.be (S.J. Husson).

Abbreviations: Da, Dalton; flp, FMRFamide-like peptide; m/z, massonization time-of-flight mass spectrometry; MS/MS, tandem mass spe196-9781/$ – see front matter # 2008 Elsevier Inc. All rights reservedoi:10.1016/j.peptides.2008.07.021

encodes for over 22,000 proteins and is 1/30th the size of the

human genome. Caenorhabditis briggsae is the closest nema-

tode species to C. elegans that shares the hermaphroditic mode

of reproduction. Although these Caenorhabditis species

diverged roughly 100 million years ago, they have almost

identical morphology and are thus only distinguishable from

each other by an experienced eye. This ‘‘cousin’’ was selected

for genome sequencing in order to improve gene annotation of

the C. elegans genome [11]. As C. briggsae is a close relative of C.

elegans, its biology and genomics were recently reviewed in the

C. elegans on-line bible, wormbook, describing resources and

genetic tools to facilitate comparative studies to infer species-

to charge ratio; MALDI-TOF MS, matrix-assisted laser desorptionctrometry; nlp, neuropeptide-like protein; TFA, trifluoroacetic acid..

p e p t i d e s 3 0 ( 2 0 0 9 ) 4 4 9 – 4 5 7450

specific function of orthologous genes [12]. About 5 years ago,

the surprisingly divergent genomic sequence ofC. briggsaewas

reported, and forms the basis for further evolutionary studies

[45]. They share the same chromosome number and genome

size that encodes roughly the same amount of proteins.

Interestingly, however, there are significant molecular differ-

ences between the two genomes. Approximately 800 C.

briggsae genes have no apparent match in C. elegans, and

nearly one third of the genome is arguably different from C.

elegans as only 65% of the genes of C. elegans orthologous genes

could be found in C. briggsae. Moreover, aligning the genomes

of the two Caenorhabditis species revealed that the intronic and

intergenic sequences are rarely conserved, while exonic

sequences appear to be highly conserved [25]. Interestingly,

the limited sequence conservation in introns flanking alter-

natively spliced exons in these two nematodes contrasts with

observations from interspecies genome alignments in mam-

mals [23]. Obviously, a better understanding of evolutionary

conservation and divergence between these two species

requires detailed studies of individual gene families. Doing

so, the functional constraint and divergence in the guanine

nucleotide-binding protein gene family has been studied in

detail [22], and more recently, a comparative genomic analysis

of the small heat shock proteins has been performed [1]. Here,

we focus on the FMRFamide-like peptide (FLP) and neuropep-

tide-like protein (NLP) gene families.

Endogenous neuropeptides are important signaling

molecules that regulate various physiological processes

and behavior in all metazoan species. Genetic, biochemical

and in silico analyses revealed 33 FLP genes, 45 NLP genes

and 40 insulin-like peptide (INS) genes in C. elegans

[17,20,26,28,29,37,38], while thus far no neuropeptide genes

are annotated in the C. briggsae genome. The synthesis of

bioactive peptides involves a series of enzymatic processing

steps. After removal of the signal peptide, the peptide

precursor is processed by proprotein convertases (PCs) at

defined cleavage motifs displaying basic amino acids. Among

the four members of the kex2/subtilisin-like proprotein

convertase (KPC) family in C. elegans [46], kpc-2/egl-3 is

exclusively expressed in the nervous system and is considered

as the major PC needed for the processing of neuropeptides in

this nematode [18,24]. In the next step of biological peptide

synthesis, a carboxypeptidase, encoded by egl-21, will remove

the basic residues from the carboxyterminal end of the

intermediate peptides [19,21]. Finally, the carboxyterminal

glycine, if present, will be transformed into an amide by the

peptidyl-glycine amidating monooxygenase. This is a com-

mon post-translational modification for endogenous neuro-

peptides. After biosynthesis in the endoplasmatic reticulum

and Golgi apparatus of neurons, the neuropeptides are stored

in the dense core vesicles which allow transport and release of

the messenger molecules in order to bind to G-protein coupled

receptors and initiate a signaling cascade.

Initially, biochemical analyses of neuropeptides were

hampered by difficulties in purification procedures. In the

past, the sequencing of one single neuropeptide was preceded

by heroic efforts for tissue collection, dissections and multiple

chromatographic separations to purify one biologically active

peptide. In contrast, a peptidomics approach aims to identify

these endogenous peptides using liquid chromatography (LC)

and mass spectrometry, in analogy with proteomics [4,16].

This way, the peptide complement of only two nematode

species, C. elegans [17] and Ascaris suum [49,50] have been

explored in a high-throughput manner. Here, we use an off-

line peptidomics approach combining liquid chromatography

and matrix-assisted laser desorption ionization time-of-flight

(MALDI-TOF) mass spectrometry to map the peptide profile of

C. elegans and C. briggsae.

2. Materials and methods

2.1. Strains

The wild type C. elegans strain N2 and the C. briggsae strain

VT847 were kindly provided by the Caenorhabditis Genetics

Centre (CGC). Both nematodes were cultured at 20 8C, using

standard nematode growth media (NGM) containing a thin

layer of E. coli OP50 bacteria [6].

2.2. Extraction of endogenous peptides and samplepreparation

Neuropeptides were extracted from both Caenorhabditis strains

in exactly the same way, using a slightly modified protocol as

previously described [17]. A mixed culture of worms were

harvested from fifteen fully grown Petri dishes (diameter

90 mm) using 0.1 M NaCl. To get rid of the bacteria and dead

animals, a density gradient that was created by adding an

equal volume of 60% of sugar and centrifugation at 500 � g for

4 min. Living nematodes were recovered from the top of the

gradient and were subsequently washed using 0.1 M NaCl.

Next, the nematode pellet was placed in 5 mL of an ice cold

extraction solvent containing methanol/water/acetic acid (90/

9/1, v/v/v) on dry ice. After homogenization of the nematodes

and sonication, the resulting solution was cleared by

centrifugation and filtration (0.22 mm). The methanol was

evaporated using a Speedvac concentrator (Savant instru-

ments Inc., Farmingdale, NY) in order to re-extract the

resulting aqueous solution with ethyl acetate and n-hexane

to get rid of the lipids. Finally, the resulting extract was

subsequently desalted by solid phase extraction using an

Oasis HLB extraction cartridge (10 mg, Waters, Milford, MA)

according to the manufacture’s guidelines. Peptides were

eluted in 1 mL of 100% methanol and stored at 4 8C.

2.3. High-performance liquid chromatography (HPLC)

Just prior to HPLC analysis, peptide samples were lyophilized

and reconstituted in 500 mL of water containing 4% of CH3CN

and 0.1% trifluoroacetic acid (TFA) and filtered through a

22 mm spin down filter (Ultrafree-MC, Millipore Corporation,

Bedford, MA). An equal amount of material from both

Caenorhabditis strains was separated on a Symmetry C18

column (2.1 mm � 150 mm, 3.5 mm, Waters) using a liquid

chromatograph (Beckmann, Fullerton, CA) equipped with the

solvent module 126 and a Diode Array Detector Module 168

(Gold System). The two solvents used were as follows: A, water

containing 0.1% TFA and B, 80% CH3CN in 0.1% aqueous TFA.

First, a wash step for 10 min using 5% B (4% CH3CN in 0.1%

p e p t i d e s 3 0 ( 2 0 0 9 ) 4 4 9 – 4 5 7 451

aqueous TFA) was started, followed by a linear gradient from

5% B to 65% B (52% CH3CN in 0.1% aqueous TFA) in 60 min at a

flow-rate of 300 mL/min. Sixty fractions of 300 mL were

collected from the beginning of the gradient using an

automated fraction collector.

2.4. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)

MALDI-TOF MS experiments were performed on an Ultraflex II

instrument (Bruker Daltonics, GmbH, Bremen, Germany) in

positive ion, reflectron mode and calibrated using a standard

peptide mixture containing angiotensin II (1045.54 Da), angio-

tensin I (1295.68 Da), substance P (1346.73 Da), bombesin

(1618.82 Da), ACTH clip 1-17 (2092.08 Da) and ACTH clip 1-18

(2464.19 Da). One half of each HPLC fraction to be analyzed was

lyophilized and resuspended in 1 mL water/CH3CN/TFA (50/

49.9/0.1). The sample was subsequently transferred to the

steel target plate and mixed with 1 mL of matrix, a saturated

solution of a-cyano-4-hydroxy cinnamic acid in acetone.

Spectra were recorded in MS mode within a mass range from

m/z 500 to 4000. To generate peptide profiles, we used a home

made software program, VglP, which automatically compares

the generated peak list files with the theoretical masses of in

silico predicted neuropeptides. To gain sequence information,

ion peaks were fragmented using an optimized LIFT method.

All MS and MS/MS spectra were automatically processed

(background subtraction, smoothing and peak picking) using

the FlexAnalysis software (Bruker Daltonics) to generate peak

list files in pkl format. MS/MS Peak list files were submitted to a

‘‘Mascot’’ search (Matrix Science) against a home-made

database containing all predicted neuropeptide precursors

from C. elegans or C. briggsae.

2.5. In silico identification of neuropeptide precursors

To identify putative neuropeptide precursors in C. briggsae, all

known FLP and NLP precursors of C. eleganswere used as query

sequences for BLASTP analysis with default parameters. The

top results that showed a significant degree of similarity were

further manually inspected to see if the typical hallmarks of a

neuropeptide precursor are present, i.e. basic or dibasic

cleavage sites and the presence of a signal peptide (e-values

present in Supplementary Tables 1 and 2). SignalP 3.0 [5] was

used to predict the signal peptide of the novel putative

neuropeptide precursors.

Fig. 1 – HPLC chromatograms of peptide extracts from C.

briggsae and C. elegans. Peptide extracts from C. briggsae (A)

and C. elegans (B) were separated using a Symmetry C18

column (2.1 mm T 150 mm, 3.5 mm, Waters). After a wash

step for 10 min using 4% CH3CN in 0.1% aqueous TFA at a

flow-rate of 300 mL/min, a linear gradient to 52% CH3CN in

0.1% aqueous TFA in 60 min was initiated. For clarity, the

corresponding chromatogram as monitored by UV

absorption at 214 nm is shown from 30 min to the end of

the gradient as no peptides eluted in the first 20 min of the

gradient.

3. Results

3.1. Identification of neuropeptide precursors from C.briggsae

In total, 27 flp neuropeptide genes and 31 nlp genes from C.

briggsae could be found by BLAST analysis using all known FLP

and NLP precursors of C. elegans as query sequences. The flp-29,

flp-30 and flp-31 genes that were predicted in other nematodes

[35,36] do not exist in either C. elegans or C. briggsae. Not all

peptide precursors from C. elegans revealed a clear C. briggsae

homologue, such as FLP-21, FLP-22, FLP-23, NLP-22, NLP-24

through NLP-34, NLP-39 and NLP-45. In addition, some C.

briggsae precursors also appear to be wrongly or incompletely

predicted, as only a partial or no signal peptide sequence is

present. This was the case for Cb_FLP-32, Cb_NLP-15 and

Cb_NLP-38. The last exon of the flp-21gene fromC. elegans codes

for SMKRGLGPRPLRFGstop, containing the neuropeptide

GLGPRPLRFamide when fully processed. This exon and thus

this peptide, however, does not appear to present in the C.

briggsaehomologue CBG20591 (BLASTP score 2.2e�20). Also, the

predicted peptides from the NLP-4 precursor ofC. elegansdo not

seem to be present in C. briggsae, although a clear homologue,

CBG02225 (BLASTP score 1.3e�31) could be found. Most likely,

the last exons have been overlooked when annotating the C.

briggsae genes. This was also the case for Cb_NLP-38 (see next

paragraph). The putative FLP and NLP precursors from C.

briggsae are listed in Supplementary Table 1 and 2, respectively.

3.2. Comparative peptidomics of C. elegans versus C.briggsae by LC–MALDI-TOF MS

Peptide extracts from wild type C. elegans and wild type C.

briggsae underwent a one dimensional HPLC separation. The

resulting chromatograms, shown in Fig. 1A and B, indicate a

p e p t i d e s 3 0 ( 2 0 0 9 ) 4 4 9 – 4 5 7452

quite similar amount of material and complexity of the

samples. As the 15 plates of C. briggsae animals yielded slightly

less biomass of living nematodes than theC. elegans culture, the

volumes of the peptide extracts to be analyzed were adjusted

accordingly. Only fractions 20 (at 30 min) to 60 (at 70 min) were

screened by MALDI-TOF MS as no neuropeptides were ever

detected in the first fractions of the separation, which is

consistent with the UV trace of the chromatograms. As an

example, fraction 51 of bothC. briggsae andC. elegans are shown

in Fig. 2A and B, respectively. In the mass spectrum from C.

elegans, two ion peaks with m/z 1863.99 and 1912.04 were

fragmented and could be identified as GGAGEPLAFSPDMLSLR-

Famide from FLP-26 and AVVSGYDNIYQVLAPRF from NLP-35,

respectively (Fig. 2B). These neuropeptides were initially over-

looked in previous bioinformatics studies and were sequenced

for the first time in a two-dimensional liquid chromatography

(2D-nanoLC) MS/MS peptidomics setup using a quadrupole

time-of-flight (Q-TOF) instrument [17]. Interestingly, fragmen-

tation of the ion atm/z 1879.98 revealed the oxidized form of the

first peptide sequence. Fragmentation of ions at m/z 1869.93,

1877.94 and 1893.93 from fraction 51 of the peptide extract of C.

briggsae revealed the orthologous neuropeptide sequences

AGVSGYDNIYQVLAPRF (Cb_NLP-35, 1868.94 Da), AGAGE-

PLAFSPDMLSLRFamide (Cb_FLP-26, 1876.95 Da, Fig. 2C) and

Fig. 2 – MALDI-TOF MS and MS/MS output from C. briggsae and

separations of peptide extracts from C. briggsae and C. elegans we

an example, the zoom regions from m/z 1800 to 2500 of fraction

Fragmentation of abundant ion peaks could confirm the neurop

peptides from Cb_FLP-26 (C) and Cb_NLP-37 (D) are shown.

the oxidized form AGAGEPLAFSPDM(ox)LSLRFamide, respec-

tively. In addition, the peptide NNAEVVNHLLKNFGTLDRLGD-

Vamide (2436.29 Da, Fig. 2D) from the Cb_NLP-37 precursor

and SSMISPSYQFEDALGLSDALERAamide (2485.18 Da) from

Cb_NLP-11 could also be sequenced, while the slightly different

NLP-37 and NLP-11 peptides fromC. elegans, NNAEVVNHILKNF-

GALDRLGDVamide and SPAISPAYQFENAFGLSEALERAamide,

respectively, were found in adjacent fractions.

When comparing the mass spectra from C. briggsae (Fig. 3A)

with their counterparts in C. elegans (Fig. 3B), an abundant ion

peak at m/z 1412.71 could be observed in both Caenorhabditis

species. Fragmentation of the two ions yielded the same

sequence, SPAQWQRANGLWamide (Fig. 3C), containing a

typical W(X)6Wamide motif that is also present in the

myoinhibiting peptides (MIPs) from insects. This peptide

was missed in the initial in silico predictions of C. elegans

neuropeptides [37], but could be fully sequenced by Q-TOF MS/

MS [17]. Although this peptide did not appear to be present in

the predicted C. briggsae homologue CBG20435 due to wrong in

silico splicing, the MIP peptide is now proven to be present in C.

briggsae by mass spectrometry. In total, this off-line HPLC–

MALDI-TOF MS setup yielded 37 FLP and 22 NLP peptides from

C. briggsae, while 37 FLP peptides and 23 NLP peptides could be

monitored in C. elegans (Table 1).

C. elegans extracts. Fractions 20–60 of the chromatographic

re analyzed by MALDI-TOF MS and pair wise compared. As

51 from both C. briggsae (A) and C. elegans (B) are shown.

eptide sequences as indicated. MS/MS spectra of C. briggsae

Fig. 3 – Biochemical evidence for the MIP/Cb_NLP-38

peptide SPAQWQRANGLWamide in C. briggsae.

Comparison of the MALDI-TOF mass spectra from fraction

43 of C. briggsae (A) and C. elegans (B) revealed an abundant

ion peak at m/z 1412.71 in both Caenorhabditis species.

Fragmentation of the two ions yielded the same sequence,

containing a typical W(X)6Wamide motif as present in the

myoinhibiting peptides (MIPs) from insects. MS/MS

spectrum of the C. briggsae peptide

SPAQWQRANGLWamide is shown in (C).

p e p t i d e s 3 0 ( 2 0 0 9 ) 4 4 9 – 4 5 7 453

4. Discussion

Dissecting neuronal circuits and linking molecular players to

animals’ behavior has often been hindered by the absence of

primary sequence information of the biochemical substrates

such as neuropeptides and their G-protein coupled receptors.

The progress of various genome sequencing projects has

opened the door for numerous in silico and peptidomic

analyses to characterize neuropeptide-encoding genes. This

way, genes from the fruit fly Drosophila melanogaster

[2,3,13,31,48], the malaria mosquito Anopheles gambiae [41],

the honey bee Apis mellifera [15] and the red flour beetle

Tribolium castaneum [27] were annotated as neuropeptide-

encoding genes. Using bioinformatics, three neuropeptide

gene groups could be found in the nematode C. elegans. Li and

coworkers identified 24 FMRFamide like peptide (flp) genes

(flp-1 to flp-23 and flp-28) encoding peptides with the common

RFamide motif at the carboxyterminus [26,29,30]. This

neuropeptide family appears to be highly represented in

nematodes [35,36]. Nathoo et al. introduced the so-called

neuropeptide-like protein (nlp) gene family, containing

encoded neuropeptide sequences that show sequence resem-

blance with other invertebrate neuropeptides such as the

sulfakinins, orcokinins, allatostatins, myomodulins, etc. [37].

Additional nlp genes that encode peptides displaying sequence

resemblance with leucokinins and the cardioactive or cardi-

oacceleratory peptides (CAPs or CAPAs) could later on be found

by BLASTP [20]. Using a comprehensive bioinformatics study,

insulin-like genes were also found in the C. elegans genome

[9,38]. From this information, however, one cannot deduce

whether all these putative peptides are actually present. The

biosynthesis of neuropeptides is regulated at different levels;

from gene expression to the actual processing of the

precursor. Obviously, this cascade of events takes place at

different places in the cell (nucleus, ER, Golgi apparatus) and

this happens at specific times. As the biosynthesis of a

neuropeptide is highly regulated in time and space, wet-lab

experiments are required to validate the in silico predictions

and to characterize the individual neuropeptides in a

biochemical way.

An initial peptidomics study revealed about 60 neuropep-

tides in C. elegans [17]. Using a quadrupole time-of-flight mass

spectrometer (Q-TOF MS), we were able to sequence 11 novel

endogenous peptides, which originate from 9 novel precur-

sors. A different peptidomics approach in which the peptide

contents of HPLC fractions are monitored using a MALDI-TOF

instrument allowed a robust comparison of neuropeptide

profiles from C. elegans strains having mutations in the

presumed peptide precursor processing enzymes. This

resulted in the characterization of the major processing

enzymes KPC-2/EGL-3 [18] and CPE/EGL-21 [19] in C. elegans.

Here, a similar differential peptidomics approach was used to

explore the peptidome of its ‘‘cousin’’, the nematode C.

briggsae. Mass spectra from each HPLC fraction from both

Caenorhabditis species can be compared pair-wise, as no (or

very little) shift in retention time appears to be present. This

can be concluded from the fact that the same neuropeptides

are present in the same fraction from both strains. The

presence of small uncharacterized ion signals at m/z 1851.11,

2064.04 and 2078.12 in fraction 51 from both C. briggsae (Fig. 2A)

Table 1 – Neuropeptide profile of C. briggsae and C. elegans as analyzed by LC–MALDI-TOF MS

Caenorhabditis briggsae Caenorhabditis elegans

Gene Peptide sequence Error Gene Peptide sequence Error

Cb_nlp-1 VNLDPNSFRMSFa 0.00 Ce_nlp-1 VNLDPNSFRMSFa �0.01

Cb_nlp-6 APKQMVRFa 0.02

Cb_nlp-6 ASMRSFNMGFa 0.03

Cb_nlp-6 YKPRSFAMGFa 0.01 Ce_nlp-6 YKPRSFAMGFa 0.00

Cb_nlp-7 GSDIDDPRFFSGAFa �0.01

Ce_nlp-7 MILPSLADLHRYTMYD 0.05

Cb_nlp-8 YPYLIFPASPSSGDSRRLV �0.03 Ce_nlp-8 YPYLIFPASPSSGDSRRLV 0.05

Cb_nlp-8 FDRYEEENPYGYNFGAHIF �0.02

Cb_nlp-8 AFDRFDNSGVFSFGS �0.01 Ce_nlp-8 AFDRFDNSGVFSFGA 0.03

Cb_nlp-8 SADPYRFMTVPT 0.00

Cb_nlp-9 GGGRAFNHNANLFRYE 0.02

Cb_nlp-11 SSMISPSYQFEDALGLSDALERAa �0.01 Ce_nlp-11 SPAISPAYQFENAFGLSEALERAa 0.07

Ce_nlp-11 HISPSYDVEIDAGNMRNLLDIa 0.08

Ce_nlp-11 SAPMASDYGNQFQMYNRLIDAa 0.06

Cb_nlp-12 TQSPTFDRQD 0.02

Cb_nlp-12 DYRPLQFa 0.01 Ce_nlp-12 DYRPLQFa 0.03

Cb_nlp-12 DGYRPLQFa 0.02 Ce_nlp-12 DGYRPLQFamide 0.03

Ce_nlp-13 AEDYERQIMAFa 0.01

Ce_nlp-13 SPVDYDRPIMAFa 0.02

Ce_nlp-13 SAPSDFSRDIMSFa 0.03

Ce_nlp-16 NAEDHHEHQ 0.01

Cb_nlp-18 SPYRTFAFA 0.01 Ce_nlp-18 SPYRTFAFA 0.03

Cb_nlp-19 MGMRLPNIIFL 0.00

Cb_nlp-19 IGLRLPNML �0.01

Cb_nlp-21 GGGRAFYDE 0.02 Ce_nlp-21 GGARAFYDE �0.03

Cb_nlp-35 AGVSGYDNIYQVLAPRF �0.01 Ce_nlp-35 AVVSGYDNIYQVLAPRF 0.04

Cb_nlp-37 NNAEVVNHLLKNFGTLDRLGDVa 0.01 Ce_nlp-37 NNAEVVNHILKNFGALDRLGDVa 0.08

Cb_nlp-38 TPQNWNKLNSLWa 0.01 Ce_nlp-38 TPQNWNKLNSLWa 0.05

Cb_nlp-38 SPAQWQRANGLWa 0.00 Ce_nlp-38 SPAQWQRANGLWa 0.01

Ce_nlp-38 ASDDRVLGWNKAHGLWa 0.01

Ce_nlp-40 APSAPAGLEEKLR 0.00

Ce_nlp-41 APGLFELPSRSVRLI 0.04

Cb_flp-1 SDPNFLRFa 0.01 Ce_flp-1 SDPNFLRFa 0.04

Cb_flp-1 SADPNFLRFa 0.02 Ce_flp-1 SADPNFLRFa 0.02

Cb_flp-1 AGSDPNFLRFa 0.01 Ce_flp-1 AGSDPNFLRFa 0.02

Cb_flp-1 AAADPNFLRFa 0.01

Cb_flp-1 SQPNFLRFa 0.02 Ce_flp-1 SQPNFLRFa 0.03

Cb_flp-2 SPREPIRFa 0.01 Ce_flp-2 SPREPIRFa 0.01

Ce_flp-3 SADDSAPFGTMRFa 0.04

Ce_flp-3 NPENDTPFGTMRFa 0.02

Cb_flp-4 ASPSFIRFa 0.02 Ce_flp-4 ASPSFIRFa �0.03

Cb_flp-5 GAKFIRFa 0.03

Cb_flp-5 AGAKFIRFa 0.02 Ce_flp-5 AGAKFIRFa 0.01

Cb_flp-5 APKPKFIRFa 0.01 Ce_flp-5 APKPKFIRFa 0.01

Ce_flp-6 KSAYMRFa 0.02

Cb_flp-9 KPSFVRFa 0.02 Cb_flp-9 KPSFVRFa 0.01

Cb_flp-11 NGAPQPFVRFa 0.00 Ce_flp-11 NGAPQPFVRFa 0.01

Cb_flp-11 ASGGMRNALVRFa 0.00 Ce_flp-11 ASGGMRNALVRFa 0.01

Cb_flp-12 RNKFEFIRFa 0.01 Ce_flp-12 RNKFEFIRFa 0.02

Cb_flp-13 AADGAPLIRFa 0.01 Ce_flp-13 AADGAPLIRFa 0.02

Cb_flp-13 ASSAPLIRFa 0.00 Ce_flp-13 ASSAPLIRFa 0.01

Cb_flp-13 APEASPFIRFa 0.01 Ce_flp-13 APEASPFIRFa 0.02

Cb_flp-13 ASPSAPLIRFa 0.01 Ce_flp-13 ASPSAPLIRFa 0.03

Cb_flp-13 SPSAAPLIRFa 0.01 Ce_flp-13 SPSAVPLIRFa 0.01

Cb_flp-13 AAPSAPLIRFa 0.01

Cb_flp-14 KHEYLRFa 0.01 Ce_flp-14 KHEYLRFa 0.01

Cb_flp-15 GGPQGPLRFa 0.01 Ce_flp-15 GGPQGPLRFa 0.03

Cb_flp-15 RGPSGPLRFa 0.01 Ce_flp-15 RGPSGPLRFa 0.02

Cb_flp-16 GQTFVRFa 0.02 Ce_flp-16 GQTFVRFa 0.02

Cb_flp-16 AQTFVRFa 0.03 Ce_flp-16 AQTFVRFa 0.02

Cb_flp-18 DFDGAMPGVLRFa �0.01 Ce_flp-18 DFDGAMPGVLRFa 0.03

Cb_flp-18 EIPGVLRFa 0.02 Ce_flp-18 EIPGVLRFa 0.02

Cb_flp-18 AYFDEKKSVPGVLRFa 0.00

Cb_flp-18 SEVPGVLRFa 0.01 Ce_flp-18 SEVPGVLRFa 0.02

p e p t i d e s 3 0 ( 2 0 0 9 ) 4 4 9 – 4 5 7454

Table 1 (Continued )Caenorhabditis briggsae Caenorhabditis elegans

Gene Peptide sequence Error Gene Peptide sequence Error

Cb_flp-18 DVPGVLRFa 0.02 Ce_flp-18 DVPGVLRFa 0.02

Cb_flp-18 SVPGVLRFa 0.02 Ce_flp-18 SVPGVLRFa 0.03

Cb_flp-19 WANQVRFa 0.01 Ce_flp-19 WANQVRFa 0.02

Cb_flp-19 ASWASSVRFa 0.02 Ce_flp-19 ASWASSVRFa 0.05

Ce_flp-22 SPSAKWMRFa 0.02

Cb_flp-24 VPSAGDMMVRFa 0.01 Ce_flp-24 VPSAGDMMVRFa 0.01

Cb_flp-26 EFNADDLTLRFa �0.02 Ce_flp-26 EFNADDLTLRFa �0.01

Cb_flp-26 AGAGEPLAFSPDMLSLRFa �0.01 Ce_flp-26 GGAGEPLAFSPDMLSLRFa 0.06

Cb_flp-28 APNRVLMRFa 0.00 Ce_flp-28 APNRVLMRFa 0.02

p e p t i d e s 3 0 ( 2 0 0 9 ) 4 4 9 – 4 5 7 455

and C. elegans (Fig. 2B), for example, also indicates that the

respective mass spectra can be compared. In total, 59

neuropeptides could be biochemically monitored from C.

briggsae while 60 peptides were identified in C. elegans. Our

results indicate the presence of very similar (homologous)

neuropeptides in both strains. Especially the FLP peptides

appear to be strongly conserved. Interestingly, a defined set of

very similar FLP peptides also occur in various Ascaris suum

nervous structures [50].

In order to facilitate the interpretation of generated mass

spectra, we first performed an in silico analysis using BLASTP to

reveal the presence of orthologous neuropeptide precursors in

C. briggsae. Not all C. elegans precursors revealed a clear

homologue inC. briggsae. These observations are completely in

line with genome-wide in silico studies. About one third of the

C. briggsae genome appears to be different from C. elegans as

only 12,155 orthologous gene pairs could be found. This

corresponds to 62% of the C. briggsae genes or 65% of the gene

set from C. elegans [45]. As the C. elegans precursors NLP-24

through NLP-34 contain a lot of GGY repeats or YGGW motifs,

it is hard to find the proper C. briggsae homologues. Most of

these genes appear to be expressed in hypodermal tissue,

suggesting that they might not encode for neuropeptides,

despite mild sequence resemblance with the molluscan

peptide APGWamide [8,10,34,43,44]. Moreover, the corre-

sponding genes most likely encode antimicrobial peptides,

as at least two genes (nlp-29 and nlp-31) appear to be

differentially regulated by fungal and bacterial infections of

C. elegans [7]. In addition, no homologous peptides from these

precursors could be monitored in the A. suum central nervous

system [50].

Manual inspection of putative neuropeptide precursors

from our BLASTP output, previously annotated as ‘‘hypothe-

tical proteins’’, indicates that some precursors have been

wrongly predicted. For some precursors, no signal peptide

appears to be present, or the last exon was missed due to

incomplete in silico splicing. Although not present in the

previously (wrongly) predicted ‘‘hypothetical protein’’ that

contains striking homology with the NLP-38 precursor from C.

elegans, a myoinhibiting peptide (MIP, or allatostatin B-like

peptide) SPAQWQRANGLWamide from the Cb_NLP-38 pre-

cursor CBG20435 could be fragmented in our peptidomics

study. Interestingly, the last exon of the nlp-38 gene from C.

elegans, encoding for SPAQWQRANGLWGRstop, appears to be

present in the C. briggsae contig cb25.fpc4180 from assembly

cb25.agp8 (Genbank accession no. CAAC01000110, nucleotide

324884 to 324929). It is thus very likely that the gene encoding

the CBG20435 protein was wrongly spliced in silico, as this

contig also contains the coding sequence of the initially

predicted CBG20435 protein. MIPs were first isolated and

characterized from an extract of 9000 locust brains [42], and

later from crickets (Gryllus bimaculatus), lepidopterans (Man-

duca sexta and Bombyx mori), and the stick insect Carausius

morosus [5,14,32,33], suggesting a conserved biological activity.

In addition to a better annotation of the C. briggsae genome,

this study also provides a promising basis for evolutionary

comparisons. Most of the neuropeptide-encoding genes from

C. elegans that appear to be evolutionarily conserved among

invertebrates, such as the allatostatins (A-type, nlp-5 and nlp-6

and B-type, nlp-38 and nlp-42), sulfakinins (nlp-12), orcokinins

(nlp-14 and nlp-15), myomodulins (nlp-2, nlp-22 and nlp-23),

leucokinins (nlp-43) and the cardioactive or cardioacceleratory

peptides (CAPs or CAPAs, encoded by nlp-44), also appear to be

present in C. briggsae. CAPs or CAPAs can be grouped in the

widely occurring periviscerokinin (PVK) family of insect

neuropeptides of which most members contain a carbox-

yterminal PRVamide, PRIamide or PRLamide motif [40]. The

CAPA/NLP-44 precursor from C. elegans contains the two

peptides APHPSSALLVPYPRVamide and AFFYTPRIamide,

while the Cb_NLP-44 orthologue encodes the same peptide

APHPSSALLVPYPRVamide in addition to AFFYAPRVamide,

which has another carboxyterminal motif. Interestingly, the

Stinkbugs Nezara viridula, Acrosternum hilare and Banasa dimiata

all contain two CAPA-PVK peptides displaying the PRVamide

motif and one CAPA-PK having a PRLamide motif, while the

Brown Stinkbug Euschistus servus contains (almost) the same

three peptides from which one displays the PRIamide motif

instead of the PRVamide sequence, as analyzed by MALDI-TOF

MS [39]. The latter species also expresses highly modified

pyrokinins. These results indicated that peptidomics studies

can provide a better understanding of evolutionary or

comparative neuroendocrinology.

Here we showed that the well-established genomic

information of a model organism such as C. elegans, can be

efficiently used to facilitate the annotation of particular gene

families of a closely related species. Doing so, we were able to

explore the peptidome ofC. briggsaeby in silico and biochemical

strategies. Obviously, this approach can be of particular use to

gain insights in the peptide profile of other (parasitic)

nematode species. As neuropeptides are important signaling

molecules that regulate many typical behaviors like food

intake, egg-laying, locomotion, etc., these endogenous bioac-

tive entities and their receptors can be considered as

important drug targets. In this context, peptidomics strategies

p e p t i d e s 3 0 ( 2 0 0 9 ) 4 4 9 – 4 5 7456

and the resulting peptide sequences might be useful to design

anthelmintic drugs.

Acknowledgements

This project was sponsored by the Research Foundation

Flanders (FWO-Vlaanderen grant G.0434.07 and 1.5.137.06).

The authors strongly acknowledge the Interfacultary Centre

for Proteomics and Metabolomics ‘‘Prometa’’, K.U.Leuven and

wish to thank the Caenorhabditis Genetics Centre for providing

the C. elegans and C. briggsae strains. We also want to thank

Johan Temmerman for writing the VglP software. S.J. Husson

and E. Clynen are postdoctoral fellows of the Research

Foundation Flanders (FWO-Vlaanderen) and T. Janssen was

supported by a Ph.D. scholarship of the Research Foundation

Flanders. K. Boonen and M. Lindemans benefit from a Ph.D.

grant of the Institute for Promotion of Innovation through

Science and Technology in Flanders (IWT-Vlaanderen), while

B. Landuyt is a postdoctoral fellow of the IWT-Vlaanderen.

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.peptides.

2008.07.021.

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