Mass spectrometric investigation of the neuropeptide ... - CiteSeerX

Journal of Neurochemistry, 2003, 87, 642–656

doi:10.1046/j.1471-4159.2003.02031.x

Mass spectrometric investigation of the neuropeptide complement and release in the pericardial organs of the crab, Cancer borealis Lingjun Li,*, ,à Wayne P. Kelley, ,1 Cyrus P. Billimoria,à Andrew E. Christie,§,2 Stefan R. Pulver,à Jonathan V. Sweedler and Eve Marderà *School of Pharmacy & Department of Chemistry, University of Wisconsin, Madison, Wisconsin, USA Department of Chemistry and the Beckman Institute, University of Illinois, Urbana, Illinois, USA àDepartment of Biology, Volen Center, Brandeis University, Waltham, Massachusetts, USA §Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA

Abstract The crustacean stomatogastric ganglion (STG) is modulated by both locally released neuroactive compounds and circulating hormones. This study presents mass spectrometric characterization of the complement of peptide hormones present in one of the major neurosecretory structures, the pericardial organs (POs), and the detection of neurohormones released from the POs. Direct peptide profiling of Cancer borealis PO tissues using matrix-assisted laser desorption/ ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) revealed many previously identified peptides, including proctolin, red pigment concentrating hormone (RPCH), crustacean cardioactive peptide (CCAP), several orcokinins, and SDRNFLRFamide. This technique also detected corazonin, a well-known insect hormone, in the POs for the first time.

However, most mass spectral peaks did not correspond to previously known peptides. To characterize and identify these novel peptides, we performed MALDI postsource decay (PSD) and electrospray ionization (ESI) MS/MS de novo sequencing of peptides fractionated from PO extracts. We characterized a truncated form of previously identified TNRNFLRFamide, NRNFLRFamide. In addition, we sequenced five other novel peptides sharing a common C-terminus of RYamide from the PO tissue extracts. High K+ depolarization of isolated POs released many peptides present in this tissue, including several of the novel peptides sequenced in the current study. Keywords: Cancer borealis, MALDI MS, neuropeptides, neurosecretion, pericardial organs, postsource decay peptide sequencing. J. Neurochem. (2003) 87, 642–656.

The crustacean stomatogastric ganglion (STG) is one of the leading systems for studying the neural basis of motor pattern generation and the modulatory effects of neuroactive substances at the cellular and network levels (Marder and Hooper 1985; Harris-Warrick et al. 1992; Marder et al. 1995; Marder and Calabrese 1996; Skiebe 2001; Nusbaum

and Beenhakker 2002). Previous studies have shown that the STG is modulated by both neuroactive agents released locally from input axons and circulating hormones delivered via the hemolymph (Marder 1987; Christie et al. 1995). A large number of studies have documented that a diverse assortment of small molecule transmitters, amines and

Received April 30, 2003; revised manuscript received July 16, 2003; accepted July 16, 2003. Address correspondence and reprint requests to Dr Lingjun Li, School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705–2222, USA. E-mail: [email protected] 1 Current address: Pharmaceutical Development, GlaxoSmithKline, King of Prussia, PA 19406, USA. 2 Current address: Department of Biology, University of Washington, Seattle, WA 98195–1800, USA. Abbreviations used: ACN, acetonitrile; AST, allatostatin; CabTRP, Cancer borealis tachykinin-related peptide; CCAP, crustacean cardioactive peptide; CHH, crustacean hyperglycemic hormone; CID, colli-

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sional induced dissociation; DHB, 2,5-dihydroxybenzoic acid; DG neuron, dorsal gastric neuron; dgn, dorsal gastric nerve; ESI, electrospray ionization; IC neuron, inferior cardiac neuron; lvn, lateral ventricular nerve; LP neuron, lateral pyloric neuron; MALDI, matrixassisted laser desorption/ionization; MS, mass spectrometry; mvn, medial ventricular nerve; PD neuron, pyloric dilator neuron; pdn, pyloric dilator nerve; PO, pericardial organ; PSD, postsource decay; PY neuron, pyloric neuron; pyn, pyloric nerve; RPCH, red pigment concentrating hormone; RP-HPLC, reverse phase high performance liquid chromatography; STG, stomatogastric ganglion; STNS, stomatogastric nervous system; TFA, trifluoroacetic acid; TOF, time-of-flight; VD neuron, ventricular dilator neuron.

2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 642–656

Neuropeptides in C. borealis pericardial organs

neuropeptides are present in the STG neuropil. Several recent reports have shown that many of the same neuromodulatory compounds are also present in the neurohemal organs of crustaceans (Christie et al. 1995; Skiebe 1999; Skiebe et al. 1999; Pulver and Marder 2002). One such neurohemal structure, the pericardial organ (PO), has long been known to be an important source of circulating hormones. Moreover, several studies have shown that many of the hormones present in the POs can modulate the neural circuits in the STG, which is situated in the ophthalmic artery anterior to the heart (Alexandrowicz and Carlisle 1953; Stangier et al. 1986; Stangier et al. 1988; Keller 1992; Christie and Nusbaum 1995). The current study focuses on mass spectrometric characterization of neuropeptides present in and released from the POs of the crab Cancer borealis. Most of our knowledge concerning the peptide content of the stomatogastric nervous system (STNS) and the POs is based on immunocytochemistry. This technique, while a powerful first step in determining modulator presence, leaves the amino acid sequence identity of the native peptides unknown. Clearly, unambiguous determination of the actual peptide structure with accurate molecular weight and amino acid sequence information is essential for studies of the physiological functions of these molecules. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is a highly accurate method that can be used to directly profile peptides from complex biological samples such as tissue sections or single cells with minimal sample preparation (Jimenez et al. 1994; Li et al. 1999b; Li et al. 2000a; Li et al. 2000b; Predel 2001). Its application to peptide identification has recently been extended to the crustacean STNS in the identification and characterization of multiple members of the orcokinin peptide family (Li et al. 2002; Skiebe et al. 2002). In the current study, we aim to determine the complement of peptides present in the POs of the crab, C. borealis. To overcome the challenges of the high chemical complexity of these neuropeptides, and the almost complete lack of genomic information, we employed front-end multistage microseparation to simplify cellular matrices, and performed de novo mass spectrometric sequencing to obtain the primary structures of several previously unknown peptides. To explore the putative hormonal role of these peptides, we also performed the first mass spectrometric assay of peptide release from the POs using high K+-induced depolarizations. Materials and methods Animals and dissection Jonah’s crabs, C. borealis were obtained from Commercial Lobster (Boston, MA, USA) and maintained without food in artificial seawater tanks at 10–12C. Animals were cold-anesthetized by packing in ice for 15–30 min prior to dissection. The POs were dissected by removing the carapace covering the heart and

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subsequently removing the longitudinal body muscles, hyperdermis, and heart. The animal was then pinned ventral side up in a Sylgardlined dissection dish to expose the pericardial cavity. The POs were identified visually as an iridescent web of nerves branching over the muscles surrounding the pericardial cavity and dissected free. All dissection was carried out in chilled (approximately 4C) physiological saline (composition in mM: NaCl, 440; KCl, 11; MgCl2, 26; CaCl2, 13; Trizma base, 11; maleic acid, 5; pH 7.45). Cellular sample preparation for MALDI MS analysis Small pieces of tissues were dissected and prepared for MALDI MS analysis. Physiological saline was replaced with an aqueous MALDI matrix solution, 10 mg/mL of 2,5-dihydroxybenzoic acid (DHB) (ICN Pharmaceuticals, Costa Mesa, CA, USA), to remove the extracellular salts associated with the tissue sample (Garden et al. 1996). Tungsten needles and fine forceps were used to dissect and transfer small pieces of tissue onto a MALDI sample plate containing 0.5 lL of either regular aqueous DHB (10 mg/mL) matrix solution or concentrated DHB (50 mg/mL) in acetone/water (4 : 1) mixed solvent. Once on the sample plate, the tissue was smashed with dissection tools and allowed to dry at ambient temperature and then subjected to MALDI MS analysis. Microbore reverse phase (RP)-HPLC of homogenates The extraction and first stage separation procedure were performed as reported previously (Li et al. 2002). Peptides were initially extracted from 26 POs using acidified acetone (1 : 40 : 6, concentrated HCl/acetone/H2O) as described previously (Floyd et al. 1999). Briefly, samples were homogenized in a microhomogenizer (Jencons Scientific Ltd, UK), and the supernatant drawn off and centrifuged (Baxter Biofuge 15, Mcgraw Park, IL, USA). This process was repeated several times, water was added and then the extract was concentrated under a stream of nitrogen to approximately 300 lL. Separations were performed utilizing a microbore HPLC (Magic 2002, Michrom BioResources, Auburn, CA, USA). For the first stage separation, an aliquot of the extract was injected onto a reverse phase 1.0 · 150 mm C-18 column (Reliasil) with a 5-lm particle size and 30 nm pore size. The column was equilibrated with solvent A at a programmed temperature of 35C. An aliquot of the aqueous extract was injected onto the column at a constant flow rate of 50 lL/min and a gradient developed from 5 to 98% of solvent B in 34 min. Solvent A consisted of 2% acetonitrile (ACN), 98% H2O and 0.1% trifluoroacetic acid (TFA) (v/v). Solvent B consisted of 95% ACN, 5% H2O and 0.1% TFA (v/v). Sample peaks were detected via absorbance at 214 and 280 nm wavelengths and the eluent collected by a small volume fraction collection system (Gilson FC 203B, Middleton, WI, USA). To identify peptides of interest, each fraction was screened using MALDI MS; approximately 0.25 lL of each fraction (approximately 20–200 lL total) was deposited on a MALDI MS sample plate followed by the same volume of an a-cyano-4-hydroxycinnamic acid matrix (10 mg/mL in 6 : 3 : 1 ACN/H2O/3% TFA) (Aldrich, Milwaukee, WI, USA). Thus more than 95% of each fraction was available for further assays. Second stage microbore HPLC separation Fractions assayed by MALDI time-of-flight (TOF) MS that contained insufficient intensity or interfering peaks from coelution


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were further purified by a second stage HPLC. Second stage separations were effected by changing the column to a 1.0 · 150 mm Vydac C-18 MS column (218MS5115, Grace Vydac, Hesperia, CA, USA) consisting of 5 lm particles with a 30-nm pore size, changing the solvent additives, slowing the relative flow rate and by flattening the gradient profile. Separations of aliquots from the combined first stage fractions were performed both with on-line electrospray ionization (ESI)-MS detection and off-line fraction collection. Both procedures utilized identical solvents and methods. Fractions were collected manually on a small volume fraction collection system. The selected fractions from the first-stage analysis were combined and concentrated to an aqueous medium using a commercial Speedvac system (Thermo-Savant, Holbrook, NY, USA). An aliquot of the sample was then loaded onto a peptide trap inline with the injection port of the microbore HPLC instrument. For HPLC-ESIMS experiment a 5-lL injection of the concentrate was used and the remainder, approximately 50 lL, was saved for the second stage separation and fraction collection. Each aliquot was injected onto the column at a uniform flow rate of 50 lL/min. The mobile phase consisted of solvent A: 95% H2O (Burdick and Jackson, Muskegon, MI, USA), 5% ACN, 0.1% acetic acid (v/v) and 0.02% TFA (v/v) (Sigma-Aldrich, Milwaukee, WI, USA) and B: 10% H2O, 90% 2 : 2 : 1 ACN, 0.1% acetic acid (v/v) and 0.014% TFA (v/v). A gradient was developed from 5 to 15% solvent B in 5 min and 15– 45% B in an additional 30 min. MALDI MS MALDI mass spectra were obtained using a Voyager DE STR (Applied Biosystems, Framingham, MA, USA) TOF mass spectrometer equipped with delayed ion extraction. A pulsed nitrogen laser (337 nm) was used as desorption/ionization source, and positive-ion mass spectra were acquired using both linear and reflectron mode. Each representative mass spectrum shown is the smoothed average of 128–256 laser pulses. Mass calibration was performed externally using a mixture of synthetic peptide standards (PE Biosystems, Framingham, MA, USA). Mass accuracy was typically better than 0.01% (Li et al. 1998). PSD analysis Semi-purified microbore LC fractions containing a peptide of interest, or 2 pmol of synthetic peptide standards (synthesized by either the Biotechnology Center at the University of Illinois or the Protein Chemistry Laboratory at the University of Pennsylvania, School of Medicine), were subjected to PSD analysis. For MALDI PSD analysis, the matrix a-cyano-4-hydroxycinnamic acid (10 mg/ mL in 6 : 3 : 1 ACN/water/3% TFA) was used. In these experiments, the total acceleration voltage was 20 kV, grid voltage set at 75%, guide wire voltage set at 0.03%, and a delay time of 75 ns used. By the use of timed ion selector, different precursor ions were selected from a mixture of peptides and subjected to fragmentation. Under these experimental conditions, the mass accuracy of the precursor ion was within 30 p.p.m., and the average error on the mass assignment of the PSD ions was less than 0.3 Da. Spectra were obtained by accumulating data from 100 to 256 laser shots. To obtain complete PSD spectra, a series of reflectron TOF spectral segments were acquired, each optimized to focus fragment ions within different m/z ranges (Kaufmann et al. 1993). Each segment

was stitched together using the Biospectrometry Workstation software to generate a composite PSD spectrum. On-line ESI MS/MS analysis For on-line MS detection, the column was connected directly to the atmospheric pressure inlet port of an LCQ Deca, ESI-ion trap mass spectrometer (Thermo-Finnigan, San Jose, CA, USA). The transfer volume to the MS detector was calculated to be similar to that of the absorbance detector resulting in retention times that are similar to the fraction collection assays. The MS tune method employed a spray voltage of 4.3 kV, a capillary temperature of 220C, capillary voltage of 21 V and a tube lens offset of 10 V. By default, automatic gain was used to control injection of ions into the trap. The Xcalibur instrument setup software (Thermo-Finnigan, San Jose, CA, USA) employed a modified triple play data dependent acquisition control. Briefly, a full scan MS was followed by a zoomscan and then MS2 of the largest peak in the full scan. Dynamic exclusion was utilized in order to include high resolution and MS2 information on secondary peaks within the scan. Nanospray ESI MS/MS To perform collisional-induced dissociation (CID) experiments for sequence information while utilizing minimal sample, the ion trap instrument was fitted with a static nanospray source (ThermoFinnigan, San Jose, CA, USA). An aliquot of approximately 5 lL was taken from a fraction from the second stage separation corresponding to the mass of interest, concentrated via Speedvac and reconstituted in 10 lL of 50/50 (v/v) CH3OH and H2O with 0.1% formic acid. The sample was loaded into a tapered, platinum coated, borosilicate nanospray emitter, PicoTip (New Objective, Woburn, MA, USA) and a spray voltage of 1.5 kV was applied. The instrument capillary temperature was set at 200C with an applied potential of 39 V. The optimized spray voltage and ion optic settings were adjusted using the instrument’s auto tune function. Once a full scan was observed in real time, MS2 and MS3 experiments were directed by manual input of the m/z-values. Tandem MS sequencing experiments used a mass isolation width of 1.5 Da for the precursor ion and 1.0 Da for resulting MS2 fragment ions to be retained in the trap. An activation Q (ion instability parameter) value of 0.25 at an activation time of 30 ms was utilized. The normalized collisional (RF) energy was set at 35% for both MS2 and MS3 experiments. Spectra were acquired for approximately 2 min resulting in 164 scans. Sequence verification was facilitated using the online Protein Prospector, MS-Product program from the University of California, San Francisco. MALDI assay for peptides released from the POs To depolarize the POs, we employed a 10-fold higher concentration of K+ (110 mM) than that in normal saline to the isolated pair of POs from each animal (N ¼ 3). A cocktail of peptidase inhibitors including amastatin (20 lM), leupeptin (10 lM), antipain-dihydrochloride (71 lM), bestatin (130 lM), phosphoramidon (37 lM), and aprotinin (0.3 lM) (Roche Molecular Biochemicals, Germany) was used throughout the experiment. All of the preparations were kept on ice (4C) for the duration of the experiment. To determine if the detected peptides were released in a depolarization and Ca2+dependent manner, we repeated the experiment without high K+ depolarization in normal saline as well as in high K+/low Ca2+



(1.3 mM Ca2+ and 13 mM Mn2+) saline. Each preparation was incubated for 10 min in the experimental condition with two washes in normal saline between each condition to remove peptides resulting from previous experimental protocol. We sampled 10 lL of releasate from each experimental condition, desalted and concentrated the releasate with ZipTip pipette tips (Millipore, Bellerica, MA, USA) packed with C18 reverse phase media prior to spotting onto the MALDI sample plate. Briefly, the ZipTip was wet with 50% ACN in Milli-Q water, equilibrated with 0.1% TFA in Milli-Q water, loaded with sample containing 0.1% TFA by aspirating and dispensing the sample 10 times, and then washed with 5% methanol in 0.1% TFA/water, followed by elution directly onto a MALDI MS sample plate with 2 lL DHB (50 mg/ mL) in 50% ACN with 0.1% TFA. Electrophysiology The stomach was removed as previously described. The STNS was isolated from the stomach and pinned onto a Sylgard (Dow Corning, Midland, MI, USA) coated dish. The STG and the stomatogastric nerve (stn) were desheathed. The STG was isolated from anterior ganglia inputs by placing isotonic sucrose (750 mM) containing 10)6 M tetrodotoxin in a Vaseline well built around the stn. Extracellular recordings were made from pyloric and gastric nerves using stainless steel pin electrodes within saline-filled Vaseline wells. Signals were amplified by an AM-Systems 1700 differential amplifier (Carlsborg, WA, USA) and recorded using an Axon Instruments (Foster City, CA, USA) data interface board. Spike time data were extracted using scripts written by Dirk Bucher in Spike2 (Cambridge Electronic Design, Cambridge, UK). Two-minute epochs of data were used in the analysis of peptide actions. The two minutes preceding the application of peptide and the eighth to tenth minute of application were analyzed.

Results

Direct MALDI MS profiling of peptides in the pericardial organs reveals a multitude of peptides present As shown in Fig. 1, direct MALDI MS profiling of a small piece of freshly isolated PO from C. borealis revealed the presence of a number of previously identified peptides including proctolin, crustacean cardioactive peptide (CCAP), SDRNFLRFa, and Ala13-orcokinin, as well as many other unknown peptides. Figure 1(b) shows the mass spectrum acquired in the high mass region. A number of peaks were detected in the mass range spanning from 2000 to 9650 Da. A multitude of CHHs have been previously characterized in sinus gland (SG) neurosecretory system in the eyestalk and POs of various species of crustacea (Keller 1992; Dircksen et al. 2001), with molecular weights ranging from 8420 to 8634 Da. The peak at m/z 8561.70, however, does not correspond to any of the previously identified CHHs. The detection of this peak and a few other peaks around 8000 and 9000 Da in the mass spectrum suggested the possible presence of CHH peptides in the crab, C. borealis. Another interesting peak that might be related to the CHH precursor was the mass spectral peak at m/z

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(a)

(b)

Fig. 1 Representative MALDI mass spectrum of a small piece of freshly isolated pericardial organ from C. borealis. (a). The mass region of 625–1525 is displayed (b). The mass range from 2000 to 9650 is displayed. Signals correspond to protonated molecular ions, [M + H]+, where M is the molecular weight of each peptide. Several previously identified peptides are labeled with asterisks, and represented in bold face.

4071.68, whose molecular weight was in close agreement to that of CHH-precursor related peptide (CPRP, with 0.002% mass measurement error) of the shore crab, Carcinus maenas (Dircksen et al. 2001). However, without additional information from sequencing analysis, conclusive assignment of these mass spectral peaks was not possible. Because peptides are distributed differentially throughout the POs, direct tissue profiling only provides a snapshot of the peptides present in a specific region of this neurohemal organ. Due to the heterogeneous distribution of modulators in this tissue, a given peptide was not always detected in the MALDI mass spectra of individual PO tissue samples. In Table 1, we consider a peptide to be present (+) in the POs only if we observed the peptide signals in over 80% of the total spectra generated (N ¼ 102). This cut-off value is determined based on the occurrence of known peptides present in the POs and the reproducibility of our MS detection. Off-line coupling microbore LC fractionation of PO tissue extract with MALDI MS detection for peptide sequencing To obtain a comprehensive description of peptides present in the PO tissue and simplify the complex mixture resulting from the tissue extract, we performed multiple stages of microbore LC separation prior to MALDI MS analysis.


Proctolin CabTRP Ib FVNSRYa FYANRYa FYSQRYa AST-3 RPCH CCAP FLRFamide-related peptide SGFYANRYa PAFYSQRYa FLRFamide-related peptide FLRFamide-related peptide Corazonin Ala13-Orcokinin Val13-Orcokinin Ser9-Orcokinin

649.36 766.38 784.41 832.41 862.42 899.44 931.43 956.39 965.54 976.46 1030.51 1053.56 1066.59 1369.63 1474.65 1502.68 1547.68

RYLPT SGFLGMRa FVNSRYa FYANRYa FYSQRYa GGSLYSFGLa pQLNFSPGWa PFCNAFTGC NRNFLRFa SGFYANRYa PAFYSQRYa SDRNFLRFa TNRNFLRFa pQTFQYSRGWTNa NFDEIDRSGFGFA NFDEIDRSGFGFV NFDEIDRSSFGFN

Peptide sequence

– – –

+ (Weimann et al. 1993) + (Weimann et al. 1993)

(Christie et al. 1995) (Christie et al. 1995) (Christie and Nusbaum 1995) (Li et al. 2002) (Li et al. 2002) (Li et al. 2002)

+ + + + + +

1995) 1995) 1995) 1995)

– – – –

al. al. al. al.

(Christie (Christie (Christie (Christie

+ + + +

et et et et

+ (Marder et al. 1986) + (Christie et al. 1997)

Previously isolated

+ (Christie et al. 1995) + (Christie et al. 1995)

Immunoreactivity + – + + – – + + + + + + – + + – –

Direct tissue MALDI* + – + + + – + + + + + + – + + + +

LC-MALDI PSD and/or ESI MS/MS

+ + + – – + – + + + + + + + + – –

Release*

*Peptides detected in direct tissue MALDI (N¼ 102) and release experiments (N¼ 12) in over 80% of the spectra are marked by a plus sign; in these two columns, peptides are identified by molecular mass measurement only, with a mass measurement accuracy of 50 p.p.m. for reflectron mode and 200 p.p.m. for linear mode. Peptide identification confirmed by MS/MS sequencing is marked with a plus sign. CCAP, crustacean cardioactive peptide; RPCH, red pigment concentrating hormone. Novel peptides identified in the current study are in bold face.

Peptide name

[M+H]+

Table 1 Peptides identified in the pericardial organs of Cancer borealis by MALDI MS

646 L. Li et al.



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A chromatogram of the first stage LC separation is depicted in Fig. 2 (center). Several representative MALDI survey spectra of corresponding LC fractions are shown surrounding the LC chromatogram. In many ways MALDI-TOF MS is an orthogonal separation technique in that it provides a further dynamic range and resolution to the separation of the crude material. As shown in the displayed MALDI spectra of various LC fractions in Fig. 2, each LC fraction still contained numerous compounds, illustrating the high complexity of the crude material. With the precursor ion selection capability, MALDI postsource decay (PSD) analysis can be performed on semipurified LC fractions to obtain peptide sequence information. As an example, Fig. 3 shows several MALDI PSD fragmentation spectra obtained from a single LC fraction, with those selected precursor ions labeled with dots. Detailed analyses of PSD fragmentation spectra of precursor ions at m/z 965.543, 976.464, 1030.51, and 1369.60 are described below; analysis of the fragmentation pattern of the precursor ion at m/z 1474.6 confirms the identity of an orcokinin peptide reported previously (Li et al. 2002).

Fig. 2 An illustration of MALDI MS tracking of microbore HPLC separation of crude tissue extract of pericardial organs from C. borealis. A chromatogram from the first stage HPLC separation of the extract is shown in the center. Detection was achieved by absorbance at a wavelength of 214 nm. Several MALDI MS survey spectra of corresponding LC fractions are displayed surrounding the LC chromatogram.

Fig. 3 MALDI PSD sequencing of multiple novel peptides from a semipurified microbore LC fraction of C. borealis pericardial organ extract. The spectrum in the center is a MALDI mass spectrum of a single LC fraction, with gray dots on the selected peaks indicating the precursor ions being isolated for MALDI PSD fragmentation. The surrounding traces are PSD composite fragmentation spectra of various precursor ions selected.

MALDI-PSD analysis of putative corazonin peptide As shown in Fig. 1, among the numerous peaks detected in the crab PO tissue, the mass spectral peak at m/z 1369.62 matches the calculated protonated molecular weight of corazonin (Mr ¼ 1369.63), an insect hormone that is conserved in all but one species examined (Veenstra 1989, 1991). To determine if the mass spectral peak at m/z 1369.62 has an identical amino acid sequence to that of authentic corazonin, we performed PSD analysis. Figure 4 shows the peptide sequence of corazonin (Fig. 4a) and PSD spectra obtained from the putative corazonin containing LC fraction (Fig. 4b, upper trace) and the synthetic corazonin standard (Fig. 4b, lower trace). The identity of authentic corazonin in the PO extract is strongly supported by the virtually identical fragmentation pattern obtained between the putative and the synthetic corazonin PSD spectra. The complete fragmentation observed from the precursor ion at m/z 1369.63 in the PO extract also allowed de novo sequencing of the peptide. Thus, we deduced the amino acid sequence of the putative corazonin peak using the mass difference between consecutive b- or y-type ions. The sequence is identical to authentic corazoninqTFQYRGWTN-amide. To confirm the sequence of corazonin, we entered the derived sequence into the MS-Product software program developed by the UCSF Mass Spectrometry Facility (http:// prospector.ucsf.edu). The software calculates the possible fragment ions resulting from PSD processes. The fragment ions detected in the PSD spectra match the predicted fragment ions and several additional internal fragment ions are labeled (Fig. 4b). The measured molecular weight of the precursor ion and the mass of the C-terminal fragment ions


648 L. Li et al.

corazonin has recently been shown to be a powerful modulator of the neural circuits present in the STG of C. borealis (A.E. Christie, unpublished observation).

(a)

(b)

Fig. 4 MALDI PSD analysis of the putative corazonin peptide. (a) The sequence of corazonin, with the N-terminal on the left, and the C-terminal on the right. Single letter amino acid abbreviations are used, with q (pGlu), T (Thr), F (Phe), Q (Gln), Y (Tyr), S (Ser), R (Arg), G (Gly), W (Trp), and N (Asn). Observed b-type (bottom) and y-type (top) ion pairs are indicated by arrows. (b) MALDI PSD fragmentation ion spectra of both the ion at m/z 1369.63 from the LC fraction of the PO extract from C. borealis (upper trace) and the synthetic corazonin standard (lower trace). As shown in the figure, the immonium ions observed in the low mass region of the spectrum (as highlighted in the inset) indicates the presence of amino acids including Ser (60), Arg (129, 70, 87, 112), Thr (74), pGlu (84), Asn (87), Gln (101), Phe (120), Tyr (136), and Trp (159). Mass signal pairs (mb + my ¼ mprecursor + 1) at m/z 112.1/1259.4, 213.2/1158.3, 360.4/1011.1, 488.5/883.0, 651.7/ 719.8, 738.8/632.7, 895.0/476.5, 952.0/419.5, 1138.2/233.2, and 1239.3/132.1 are assigned as paired b-type and y-type ions. The b-type ions are determined based on the detection of their corresponding a-type ions (loss of CO, yielding a 28-Da lower mass ion). The fragment ion labels used are based on the established nomenclature (Roepstorff and Fohlman 1984). N-Terminal ion series such as a/bions and their loss of neutrals, C-terminal ion series such as y-ions, and several internal fragment ions as well as immonium ions are labeled only in the upper trace spectrum. The masses of the fragment ion signals labeled with asterisks in the lower trace are identical to fragment ions detected in the upper trace.

confirm the amidated C-terminus and a pGlu-modified N-terminus in the peptide. This result confirms that authentic corazonin is present in the POs of C. borealis. Authentic

A new member of the FMRFamide peptide family Figure 5(a) shows the MALDI PSD fragmentation spectrum of a precursor ion at m/z 965.54. Using the same sequencing strategy outlined above, we determined the amino acid sequence to be NRNFLRFamide. This represents a truncated form of the TNRNFLRFamide peptide that was previously isolated and sequenced from the STNS of the crab C. borealis (Weimann et al. 1993). The derived amino acid sequence is shown at the top of the spectrum. The sequence interpretation was performed from both the N- (b-ions) and C-termini (y-ions). The PSD analysis of the synthetic peptide standard NRNFLRFamide produced an identical fragmentation pattern (data not shown), further substantiating the proposed sequence. The similarity between this peptide and those previously shown to be physiologically active on STG motor patterns (Weimann et al. 1993) caused us to ask whether NRNFLRFamide would mimic the actions of the previously identified extended FLRFamides. Therefore, we carried out a series of electrophysiological experiments. Figure 5(b) shows that bath application of 10)6 M synthetic NRNFLRFamide can activate both pyloric and gastric rhythms in preparations in which neither rhythm is being expressed (N ¼ 4). Shown on the left is a schematic drawing illustrating the extracellular recording sites and experimental conditions used to collect the data shown on the right. The STG was isolated from anterior ganglia inputs by placing sucrose and 10)6 M tetrodotoxin in a Vaseline well on the stn, as indicated in Fig. 5(b). Extracellular recording electrodes were placed on the nerves containing the axons of motor neurons of the STNS. The lateral ventricular nerve (lvn) carries the axons of the lateral pyloric (LP) neuron, the pyloric (PY) neurons, and pyloric dilator (PD) neurons, all active in the pyloric rhythm. The pyloric nerve (pyn) and the pyloric dilator nerve (pdn) contain axons of the PY and PD neurons, respectively. The median ventricular nerve (mvn) carries the axons of the ventricular dilator (VD) and inferior cardiac (IC) neurons. The dorsal gastric nerve (dgn) carries the axon of the dorsal gastric (DG) motor neuron, an important component of the gastric mill network. Under control conditions (Fig. 5b, right upper panel), the mvn and pdn were silent, and the other nerve recordings showed some sporadic activity. Application of 10)6 M NRNFLRFamide (Fig. 5b, right lower panel) activated the DG neuron in four of four experiments. In all four experiments the DG neuron was silent in control saline. In three of the four experiments, NRNFLRFamide induced DG bursting. In these three experiments NRNFLRFamide increased the mean number of spikes per burst from 0 to 105 ± 16.7 (standard error of the mean, p < 0.05), increased the mean burst duration from 0 to 7.91 ± 0.90 s (p < 0.02)



(a)

(b)

Fig. 5 Characterization of NRNFLRFamide. (a) MALDI PSD fragmentation spectrum of a precursor ion at m/z 965.54. The derived amino acid sequence is shown at the top of the spectrum. The fragment ions are labeled using the established nomenclature. The amino acid is deduced from both directions using the complete N- and Cterminal ion series. Detected b/y ion pairs include 115.1/851.5, 271.3/ 695.4, 385.2/581.4, 532.3/434.3, 645.3/321.2, 801.4/165.1. Immonium ions indicating the presence of Asn (87, 70), Arg (70, 87, 112, and 129), Phe (120), and Leu (86) are detected and labeled as single-letter amino acid abbreviations in Fig. 5(a). Single letter amino acid abbreviations are used, with N (Asn), R (Arg), F (Phe), and L (Leu). (b) Actions of NRNFLRFamide on the motor patterns of the stomatogastric ganglion (STG) of the crab, C. borealis. Shown on the left is a schematic diagram of the experimental configuration. The STG was isolated by placing sucrose in a Vaseline well around the stomatogastric nerve (stn). Shown on the right are simultaneous extracellular recordings from the pyloric nerve (pyn), the medial ventricular nerve (mvn), the pyloric dilator nerve (pdn), the lateral ventricular nerve (lvn), and the dorsal gastric nerve (dgn). The upper panel shows the recordings in control saline, and the lower panel shows the initiation and activation of pyloric and gastric mill rhythms upon the application of 10)6 M NRNFLRFamide.

with a mean cycle period of 16.0 s. In the fourth experiment NRNFLRFamide induced tonic firing of the DG neuron. NRNFLRFamide applications significantly increased the mean duty cycle (fraction of the time the neuron is active

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over one entire period of activity) of the LP neuron from 0.12 ± 0.05–0.23 ± 0.07 (p < 0.05). These effects are similar to those previously reported for SDRNFLRFamide and TNRNFLRFamide (Weimann et al. 1993).

De novo sequencing of RYamide peptides Because of the lack of genomic sequence information, partial sequence-specific fragmentation is not sufficient for peptide identification. Thus, only high quality PSD fragmentation spectra resulted in confident peptide identification. Several PSD analyses generated complete sequence specific fragmentation that allowed de novo sequencing of previously unknown peptides. Figure 6 shows the PSD fragmentation spectrum of a precursor ion at m/z 976.46. The determined amino acid sequence was SGFYANRY-amide. We performed the PSD analysis of the synthetic peptide of the above sequence which showed identical fragmentation, thus confirming the identity of the proposed amino acid sequence (data not shown). Another unknown peak at m/z 1030.51 in the LC fraction was also subjected to PSD fragmentation analysis as shown in Fig. 7(a). Using the same procedure outlined above, we obtained a partial sequence of P/A-FYSQRY-amide, with

Fig. 6 MALDI PSD de novo sequencing of an octapeptide (m/z 976.46) fractionated from C. borealis PO extract. The derived amino acid sequence is shown at the top of the spectrum. In the low mass region, immonium ions indicative of the presence of Gly (30), Ala (44), Ser (60), Asn (70, 87), Arg (70, 87 112), Phe (120), and Tyr (136) were detected in the spectrum. These ions are labeled using single letter code, with S (Ser), G (Gly), F (Phe), Y (Tyr), A (Ala), N (Asn), R (Arg). Starting from the high mass end, using the formula [M + H]+ )18 (H2O) –X (where X ¼ each of the 20 amino acid residue masses), the highest b-type ion is determined to be 796.4, with y1 ion at 181.1, thus indicating that the C-terminal residue is Tyr, with an amidation modification. Because the b-type ions were generally accompanied by their corresponding a-type ions in MALDI PSD fragmentation process, the b/y ion pairs were identified as following: 796.4/181.1, 640.3/337.2, 526.2/451.2, 455.2/522.3, 292.1/685.3, 145.1/832.4, 88.1/889.4. The amino acid sequence was then deduced from the mass difference between consecutive b- or y-ions. Several internal fragment ions are also labeled in the spectrum.


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Fig. 7 Mass spectrometric characterization of a novel peptide at m/z 1030.51. (a) MALDI PSD fragmentation ion spectrum of precursor ion at m/z 1030.51. The derived amino acid sequence is shown at the top of the spectrum. The detected b/y ion pairs are shown on the peptide sequence, with b-ions at the bottom and y-ions on the top. The fragment ions in the spectrum are labeled using the established nomenclature. The immonium ions are labeled with single-letter amino acid code, with P (Pro), A (Ala), F (Phe), Y (Tyr), S (Ser), Q (Gln), and R (Arg). Note the ambiguity at the N-terminal sequence. (b) MS spectrum from the HPLC-ESI MS assay corresponding to the peak from the ion chromatogram (inset) eluting at 11.3 min. The singly and doubly charged species of the m/z 1030 peak are labeled. (c) MS/MS spectrum of the singly charged m/z 1030.5 peak. Loss of H2O from the precursor was observable at m/z 1012.4. The singly charged b7 ion corresponding to the loss of RY-NH2 was readily visible at m/z 850.3. Mass spectral peaks at m/z 833.2, 832.3 and 822.3 were assigned as b7 ion with loss of NH3, H2O (not labeled) and CO (a7 ion). The low mass cut-off of the ion trap for this parent ion precluded inspection of the b2 and a2 ions; however, the corresponding y6 ion can be seen at m/z 862.3. The y6-NH3 ion was also visible. The remaining b and related ions were readily identifiable; a3/b3 (m/z 287.9 and 316.0), a4/b4 (m/z 450.9, 479.0), a5 (not labeled), b5-H2O and b5 (m/z 538.1, 548.1, 566.1), and b6-NH3 (not labeled), b6-H2O and b6 (m/z 676.1, 677.2, 694.3). Ions of singly charged y-type fragments likewise can be identified; y5 and y5-NH3 (m/z 715.3, 698.4), y4 (m/z 552.3), y3 (m/z 465.3), y2 and y2-NH3 (m/z 337.2, 320.2). The tandem MS spectrum contains the fragmentation ion information (most notably, the y7 ion at 933.5) that supports the proposed sequence of PAFYSQRY-amide.

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ambiguity of the order of the first two amino acids at the N-terminus. To obtain complementary sequence specific fragmentation information to allow the unambiguous determination of the N-terminal sequence, and to confirm the proposed sequence, we performed ESI MS/MS analysis. We pooled several fractions containing the peak at m/z 1030 from the first stage separation and performed a second stage HPLC separation (see Materials and methods). A repeat of the separation on a much smaller aliquot was performed with ESI MS detection inline. This allowed the assignment of m/zvalues for each of the fractions and rapid identification of the fraction from the larger scale separation that contained the

peak at m/z 1030.5. Figure 7(b) illustrates the ion chromatogram resulting from the HPLC-ESI MS separation (inset) and the resulting full scan MS of the peak eluting at 11.3 min. High resolution, zoomscan spectra for the two major components confirm that the peak at m/z 1030.5 was singly charged and the peak at m/z 515.82 was the doubly charged species. We used nanospray ionization (NSI) mass spectrometry to elucidate the amino acid sequence of the peak at m/z 1030.5, detected in the HPLC fraction. A MS2 collisional spectrum of the singly charged ion at 1030.5 is presented in Fig. 7(c). The tandem MS spectrum corroborated the proposed peptide sequence for the peak at m/z 1030, PAFYSQRY-amide. Compared to the MALDI PSD data, the detection of y7 ion at m/z 933.5 allowed the unambiguous assignment of the N-terminal sequence as PA. MS3 experiments on the b7 fragment were also performed (not shown) and gave similar information on the b6-b3 and y2-y5 fragments. The NSI-MS2 together with the MALDI PSD spectra and identified immonium ions provide substantive evidence for the proposed sequence. Interestingly, we also identified three other novel peptides containing RYamide at their C-terminus using the same MSbased sequencing and spectral interpretation procedure as described above. The derived amino acid sequences are FVNSRYamide, FYANRYamide, and FYSQRYamide. The



sequences of these new peptides are summarized in bold face in Table 1. We have tested the physiological actions of some of the RYamide peptides on the STG network; so far no apparent actions have been found. Study of their actions on other target tissues in C. borealis is ongoing. Peptide release from the POs To investigate the putative hormonal roles of the peptides present in the POs, we performed mass spectrometric assay

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for the peptides released upon high K+ depolarization (N ¼ 3). Figure 8 shows representative data from one experiment. Figure 8(a) shows the MALDI mass spectrum of the releasate collected in normal saline prior to high K+ depolarization. Here, a few peaks were detected, presumably due to constitutive release. Once the preparation was transferred to the high K+ saline solution, a dramatic increase in the number of peptide peaks was seen (Fig. 8b). Several of these peaks correspond to known peptides including proctolin, Cancer borealis tachykinin-related peptide Ib (CabTRP1b), CCAP, SDRNFLRFamide, and Ala13-orcokinin. In addition, we also detected many of the peptides identified for the first time in this report, including corazonin, NRNFLRFamide, FVNSRYamide, SGFYANRYamide, and PAFYSQRYamide, indicating that these new peptides are released by high K+ depolarizations. Figure 8(c) shows the result from high K+ depolarization in low Ca2+, high Mn2+ saline (reversible calcium channel blocker); minimal peptide release was observed, suggesting a Ca2+-dependence of release. Finally, after several washes with normal Ca2+ saline, we applied a second high K+ depolarization in normal saline. Many of the same peptides were released, with slightly reduced signal intensity (Fig. 8d). The last column in Table 1 summarizes the peptides detected in total of 12 release experiments. If a peptide peak was detected in over 80% of the spectra, we consider a peptide to be released (+) from the POs in response to high K+ depolarization. Due to the low concentrations of peptides being released, peptide analysis was performed in linear mode only, with average mass measurement accuracy at 200 p.p.m. Discussion

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Fig. 8 MALDI MS profiling of releasate from C. borealis PO. (a) MALDI mass spectrum of control sample in normal saline before high K+ depolarization. Baseline activity is observed with a few peaks labeled. (b) MALDI mass spectrum of releasate collected from high K+ depolarization. Numerous peptide peaks are detected, with those labeled with dots being previously known peptides or newly sequenced peptides in the current study. The inset lists all the peptides identified based on molecular weight measurement. (c) MALDI profile collected from preparation in low Ca2+, high Mn2+ (reversible calcium channel blocker) saline with high K+ depolarization. Minimal peptide release was observed. (d) MALDI mass spectrum of releasate collected from a second high K+ depolarization after switching the preparation back in normal saline. Most of the same peptides seen in (b) were detected. The identified peaks are listed in the inset.

The POs are major neurosecretory structures that can release amines and peptides into the hemolymph and elicit a variety of physiological actions. Berlind (1976) demonstrated that extracts of the POs of crabs injected into intact animals caused an increase in the frequency of scaphognathite beating. Neuromodulators found in decapod POs also modulate the amplitude and frequency of heart beat (Alexandrowicz and Carlisle 1953). Additionally, it has been shown that neuroactive substances in the POs altered properties of cardiac ganglion (Cooke and Hartline 1975), neuromuscular junctions, and muscle contractibility (Kravitz et al. 1980; Lingle 1981; Mercier et al. 1990; Meyrand and Marder 1991; Worden et al. 1995; Jorge-Rivera and Marder 1996; Jorge-Rivera et al. 1998). Furthermore, neurohormones found in the POs, are capable of exerting a wide range of modulatory effects on the neural circuits in the STG (Nusbaum and Beenhakker 2002). For all these reasons, considerable efforts have been made to determine the composition of the neuroactive substances present in the POs in various decapod species. Using immunocytochemical and biochemical techniques, a wide array of neuromodulatory


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substances have been identified in the POs in both adult animals and during embryonic and larval development (Keller 1992; Christie et al. 1995; Pulver and Marder 2002). In addition to various amines such as serotonin (Beltz and Kravitz 1983), dopamine (Siwicki et al. 1987), and octopamine (Evans et al. 1976), many peptides such as proctolin, FLRFamide-like, CCAP, and orcokinin peptides have been found in the POs of numerous decapod crustacean species (Keller 1992; Christie et al. 1995; Pulver and Marder 2002). For example, proctolin is present in the POs of the lobster, Homarus americanus (Schwarz et al. 1984), the crayfish, Procamburus clarkii (Siwicki and Bishop 1986), and the shore crab Carcinus maenas (Stangier et al. 1986). FMRFamide-like peptides are localized in the POs of the lobster H. americanus (Kobierski et al. 1987; Trimmer et al. 1987), the crayfish P. clarkii (Mercier et al. 1993), and several crab species (Krajniak 1991; Christie et al. 1995). Moreover, allatostatin-like peptides (Skiebe 1999), orcokinin family peptides (Li et al. 2002; Skiebe et al. 2002; Skiebe 2003), crustacean hyperglycemic hormone (Keller 1992; Dircksen et al. 2001), and CCAP (Stangier et al. 1988; Christie et al. 1995; Skiebe et al. 1999) have also been previously reported to be present in the POs of several decapod species. MS-based peptide identification strategy The current study represents the first mass spectrometric investigation of the neuromodulatory complement of the POs in the crab C. borealis. As summarized in Table 1, many previously identified peptides were detected in direct tissue MALDI analysis. Only four of these peptides were previously isolated and sequenced in C. borealis using conventional biochemical techniques that involved multiple steps of purification of a large pool of tissue samples followed by Edman degradation (Marder et al. 1986; Weimann et al. 1993; Christie et al. 1997). Other peptides were previously identified based only on immunoreactivities (Christie et al. 1995; Skiebe 2001; Li et al. 2002). While useful for tissue localization, immunocytochemistry suffers from limitations including cross-reactivity with structurally similar peptides thus preventing the unequivocal identification of a specific peptide, and the small number of peptides that can be analyzed simultaneously. As the next step after determining the identity of a neuromodulator in a particular neuronal circuit is often determining its physiological role using exogenous application of synthetic peptides, knowing the exact chemical structure of the putative hormone is important information not provided by immunocytochemistry. MALDI-based peptide identification allows simultaneous detection of a full spectrum of peptides present at significant concentrations directly from tissue samples with high mass accuracy. As shown in the current study, direct tissue MALDI and PSD sequencing analysis of the LC fractions resulting from the PO tissue extract confirmed the structures

of several known peptides, such as proctolin, RPCH, CCAP, extended FLRFamide-related peptides, and orcokinins. In the case of peptide families, MALDI MS analysis allows unambiguous identification of the actual forms and different members of a peptide family. For example, several forms of the extended FLRFamide-related peptides (including a novel truncated form of the FLRFamide peptide) were identified in the POs to substantiate the positive FLRFamide immunoreactivity documented previously. Similarly, Ala13-orcokinin was detected in the PO tissue samples by MALDI MS, and two additional forms of orcokinins (Val13- and Ser9-orcokinins) were detected in LC fractions from pooled PO tissue extract, indicating a possible differential expression of different forms of orcokinins in the PO, as the latter two forms of orcokinins were not observed in MALDI spectra of the tissue samples surveyed. These two forms of orcokinins are likely expressed in the different regions of the PO tissue from that of Ala13-orcokinin, or at much lower level to be detected in individual organs by MS. As we have previously demonstrated simultaneous detection of five different forms of orcokinins, including Ala13-, Val13-, and Asn13-orcokinins in the PO tissue from H. americanus (Li et al. 2002), it is unlikely that the absence of Val13- and Ser9-orcokinins is due to the difference of ionization efficiency or analyte suppression of Ala13-orcokinin. Thus, it is advantageous to use the combination of direct tissue profiling and HPLC fractionation of pooled tissue extract to generate a more complete characterization of peptides present in the POs. As demonstrated in Fig. 1, there are many mass spectral peaks that do not correspond to previously identified peptides. To characterize several of these new peptides, we performed de novo MALDI PSD sequencing of a number of unknown peaks fractionated from tissue extracts. To simplify the complex mixture resulting from the PO tissue extract and also concentrate the peaks, microbore LC separation was performed prior to MALDI MS analysis. While on-line LC coupling with ESI MS/MS has been the preferred method for large-scale peptide identification from protein digests and other complex mixtures, the off-line coupling MALDI PSD approach has some advantages, including the ability to select many more peaks for sequencing without time constraints. This is particularly useful for analyzing complex tissue extracts, where many peptides coelute and low abundance peptides are often missed in LC ESI-MS/MS analysis due to the preferential selection and identification of high-intensity peaks in the elution time window. As evident from Fig. 3, multiple PSD sequencing analyses were performed on several precursor ions from individual LC fractions, yielding enhanced chemical information from the limited amount of samples. Because no genomic sequence information is available for C. borealis, only peptides producing complete fragmentation allowed the derivation of full amino acid sequence. While all of the HPLC fractions were analyzed by mass spectrometry, many LC fractions contained larger



peptides (precursor mass > 2000 Da) that fragment less efficiently, which makes it difficult to generate complete sequence information. Furthermore, several peptides were eluted in a few consecutive LC fractions, so we were only able to fully sequence seven new peptides based on PSD fragmentation analysis, but many more peptides generated partial sequence-specific fragmentation that did not yield complete peptide identification. We have also demonstrated the use of combination of MALDI-PSD and nanoESI CID fragmentation techniques to generate complementary sequence-specific fragmentation information to allow the complete characterization of the peak at m/z 1030.51. While CID generates more efficient fragmentation, the low mass cut-off limitation associated with the ion trap mass analyzer prevents the effective observation of low mass regions. In contrast, PSD produces abundant immonium ions in the low mass region that are indicative of amino acid compositions, which are especially useful for de novo sequencing of unknown peptides. FMRFamide-like peptides FMRFamide-like peptides are perhaps the most widely distributed neuropeptides in the animal kingdom. Since their first discovery in mollusks (Price and Greenberg 1977), a large number of related peptides have been chemically characterized in many different phyla (De Loof and Schoofs 1990; Krajniak 1991; Mercier et al. 1991; Mercier et al. 1993; Schoofs et al. 1997; Li et al. 1999a; Sithigorngul et al. 2001; Baggerman et al. 2002). Two extended forms of FLRFamide, TNRNFLRFamide and SDRNFLRFamide were previously purified and sequenced in the lobster H. americanus (Trimmer et al. 1987) and the crab C. borealis (Weimann et al. 1993). Here we report a new member of the extended FLRFamide peptide, NRNFLRFamide in the C. borealis based on de novo sequencing analysis of the PO extract. This peptide was previously isolated in the POs from crayfish, P. clarkii (Mercier et al. 1993). This result is consistent with previous observation that additional FMRFamide-like immunoreactive HPLC fractions were detected in the crab nervous system (Marder et al. 1987; Weimann et al. 1993). Physiological experiments demonstrated that NRNFLRFamide elicits effects on the pyloric and gastric mill rhythms similar to those seen with TNRNFLRFamide and SDRNFLRFamide. This may suggest that only the sequence of RNFLRFamide is important for receptor recognition and binding. It is also interesting to note that NRNFLRFamide is often the most intense peak in the mass spectra of the PO tissue samples, suggesting a differential expression of these peptides in the POs and the STNS, and a potential neurohormonal role of the peptide. Corazonin While corazonin has been reported in a number of insect species (Veenstra 1989, 1991, 1994; Schoofs et al. 2000;

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Hansen et al. 2001), the occurrence of corazonin in crustacean species is documented for the first time in the current study. Consistent with the highly conserved amino acid sequence of this peptide throughout arthropod species, the de novo sequencing of the putative corazonin peak in C. borealis PO extract revealed the authentic form of this peptide is also found in C. borealis. With the availability of multiple corazonin gene and precursor sequences of several insect species, it was possible to use a multiple sequence alignment procedure to locate the highly conserved sequence. We then employed this template sequence as a guide to search for putative corazonin in crustacean species. Despite the highly conserved peptide structure, the functions of corazonin appear to be species specific and even tissue specific. For example, this peptide is highly effective at stimulating the activity of the heart and hyperneural muscle, whereas other visceral muscles are completely insensitive to this peptide (Predel and Eckert 2000). The corazonin myostimulatory effect is mainly restricted to the American cockroach, Periplaneta americana (Predel and Eckert 2000; Predel et al. 2001), while in locust His7-corazonin induces body color pigmentation (Tawfik et al. 1999; Schoofs et al. 2000). Electrophysiological experiments have demonstrated strong modulatory effects on the pyloric rhythm of the isolated STG upon application of synthetic corazonin (A.E. Christie, unpublished observation), which adds to the wide spectrum of the functions of the corazonin peptide. CHH and other larger peptides in the PO Although the major focus of the current study was characterizing peptides in the lower mass range, several peaks were detected in the mass range from 2000 to 9650 Da. One notable peptide detected in the MALDI profiling of the POs is a peak at 8562 Da, a possible candidate for CHH in C. borealis. CHH are involved in various physiological processes including regulation of blood glucose and lipids. Since the first identification of a SG-CHH in the shore crab Carcinus maenas, more than 20 SG-derived CHHs have been isolated and identified in various species of crustacea (Keller 1992). While the primary source of CHHs is in the SG in the eyestalk, multiple forms of novel CHH-like peptides were recently reported in the POs from the shore crab, C. maenas (Dircksen et al. 2001). Given the molecular masses of these previously identified CHHs, it is possible that the peak at 8562 Da might be the CHH present in C. borealis with several amino acid substitutions. Due to the inefficiency of MS/MS fragmentation for peptides larger than 2500 Da, the MS-based sequence analysis of the intact CHH peptide was not possible; therefore, the conclusive peptide assignment can not be made without additional confirmation. The detection of a mass spectral peak at m/z 4072, which corresponds to a putative peptide encoded by the CHH precursor, supported the presence of CHH in the POs of C. borealis. The identical molecular weight (and likely


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sequence) of CPRP in C. borealis to that of CPRP from C. maenas supports earlier observation that PO-CHH and SG-CHH share an identical N-terminal sequence (positions 1–40), but differ considerably in the remaining sequence. RYamides and peptide release Finally, we sequenced several new peptides sharing a common C-terminal, RYamides. While the physiological effects of these newly sequenced peptides are not known, the fact that these peptides are C-terminally amidated and released upon high K+ depolarization strongly suggests neurohormonal roles for these peptides. Several of these new RYamide peptides are coreleased with NRNFLRFamide, corazonin, proctolin, CCAP, and Ala13-orcokinin by high K+ depolarization in a Ca2+-dependent manner. This is also the first demonstration of assaying peptide release from C. borealis POs using mass spectrometric techniques. While some variability was observed between different preparations and the first and second high K+ depolarizations, several peptides including proctolin, CabTRP1b, FVNSRYamide, NRNFLRFamide, SGFYANRYamide, PAFYQSRYamide, and Ala13-ocrokinin were always released. It is interesting to note that CabTRP1b was consistently detected in releasate, yet no CabTRP1a was detected. Previous studies on the two tachykinin-related peptides showed that CabTRP1a is 20 times more abundant and 500 times more potent than CabTRP1b (Christie et al. 1997). These results led to the speculation that CabTRP1b is a breakdown product of CabTRP1a. However, the detection of CabTRP1b in releasates in the current study suggests that this shorter form of the peptide may be directly cleaved from the precursor protein and could serve a physiological role in C. borealis. In summary, the combination of both direct tissue MALDI profiling and MS-based sequencing allows comprehensive characterization of the peptide complement in a nervous system at higher throughput with greater chemical details. The large-scale mass spectrometric investigation of neuropeptides and hormones in the pericardial organs of C. borealis revealed much greater diversity and complexity of the peptide messengers than had been previously demonstrated by immunocytochemical and electrophysiological approaches. We confirmed many of the previously known peptides and unambiguously identified different chemical forms of the peptide families. Furthermore, we have fully sequenced and identified several new peptides. However, it is evident from the current study that many more peptides remain uncharacterized. It is worth noting that members of several wellknown peptide families visualized in immunocytochemical studies were not observed. This is likely due to amino acid variations from the authentic forms found in other species, post-translational modifications and perhaps cross-immunoreactivity and/or insufficient sensitivity of the current MS methods. Future work will aim to characterize these peptides by employing the combination of immunoaffinity and

MS-based sequencing approaches. Furthermore, the de novo sequencing methodology will be coupled with database searching via homology from related species whose genomic sequences are available. Such peptidomic approaches promise to significantly accelerate the discovery of new peptides and yield a complete description of the peptide signaling molecules involved in the crustacean nervous system and to further increase our understanding of peptide functions at the network level. Acknowledgements This work was supported by National Institute of Neurological Disorder and Stroke grants NS17813 (EM) and NS31609 (JVS). We thank Dr Michael Nusbaum (University of Pennsylvania School of Medicine) for the gift of synthetic corazonin.

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