Identification of Neuropeptides from the Sinus Gland of the Crayfish Orconectes limosus Using Nanoscale On-line Liquid Chromatography Tandem Mass Spectrometry*

Patrick Bulau{ddagger},§, Iris Meisen§, Tina Schmitz{ddagger}, Rainer Keller{ddagger} and Jasna Peter-Katalinic§,

From the {ddagger} Institute for Zoophysiology, University of Bonn, Endenicher Allee 11-13, D-53115 Bonn, Germany; and § Institute for Medical Physics and Biophysics, Biomedical Analytics, University of Münster, Robert-Koch-Straße 31, D-48149 Münster, Germany


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In this article, a novel and sensitive analytical strategy for direct characterization of neuropeptides from the X-Organ-sinus gland neurosecretory system of the crayfish Orconectes limosus is presented. A desalted extract corresponding to 0.5 sinus gland equivalents was analyzed in a nanoflow liquid chromatography system coupled to quadrupole time-of-flight tandem mass spectrometry (nanoLC-QTOF MS/MS). The existence and structural identity of four crustacean hyperglycemic hormone precursor-related peptide variants and two new genetic variants of the pigment-dispersing hormone, not detected by conventional chromatographic systems, molecular cloning, or immunochemical methods before, was revealed. The here-presented approach of the combined LC-QTOF MS/MS technique is a powerful tool to discover new peptide hormones in biological systems, due to its sensitivity, accuracy, and speed.


Studies in crustaceans have demonstrated the X-Organ-sinus gland neurosecretory system (XO-SG)1 as a source of various neuropeptides (1, 2). The most relevant progress has been achieved in structure elucidation of a novel family of large peptides from the sinus gland (SG), which includes, according to their first-discovered biological activities, crustacean hyperglycaemic (CHH), moult-inhibiting (MIH) and vitellogenesis/gonad-inhibiting (VIH/GIH) hormones (3, 4). Two subfamilies may be distinguished at the preprohormone level, either containing an associated CHH precursor-related peptide sequence (CPRP), located between the signal peptide and the native hormone sequence, characteristic for the CHH subfamily, or without this CPRP sequence, as determined for the MIH/VIH/GIH subfamily. CPRPs have also been identified in other invertebrate groups such as insects and nematodes (4, 5). These neurohormones are classically considered as major physiological regulators involved in the control of carbohydrate metabolism and in various functions in development such as moult and reproduction (3, 6, 7). Additionally, the chromatophore-regulating hormones, like red pigment-concentrating hormone (RPCH) and pigment-dispersing hormone (PDH), have been found in the XO-SG system of different crustacean species (811).

Different approaches, such as conventional chromatography, molecular cloning, and immunochemical methods, were used previously to investigate sinus gland components (1217). The limitations of these established methods to identify and characterize new peptides are the relatively low sensitivity in the case of conventional chromatography, the necessity of sufficient sequence homology in the case of molecular cloning, and the availability of specific antibodies in the case of immunochemical methods. In contrast to this, the combination of nanoscale liquid chromatography and mass spectrometry (MS) capable of tandem MS analysis (nanoLC-MS/MS) is designed for detection and sequencing of known and unknown peptides independent of their sequence homology. This is the first report on the direct analysis of neuropeptides from the neurosecretory XO-SG system using nanoLC-MS/MS.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals—
American freshwater crayfish, Orconectes limosus, were obtained from the Havel River in Berlin (Germany) through a commercial fisherman.

Sinus Gland Extraction and Sep-Pak Purification—
A batch of 30 SG were rapidly dissected, collected in 2 N acetic acid, and immediately frozen in liquid nitrogen. Rapid extraction of SG was carried out as described previously (18). Purification of extract was performed on a Sep-Pak C18 cartridge (Waters Corp., Milford, MA). Solvent A was composed of 0.11% trifluoroacetic acid (TFA); solvent B was composed of 0.10% TFA and 80% acetonitrile.

LC-MS/MS Analysis—
Nanoscale LC-MS analysis of SG extract aliquots corresponding to 0.5 equivalents of a SG was performed using the UltiMate capillary LC system equipped with a FAMOS autosampler (LC Packings, Amsterdam, The Netherlands) coupled to a hybrid quadrupole orthogonal acceleration time-of-flight tandem mass spectrometer (Q-TOF; Micromass, Manchester, UK). The LC-MS device was adjusted with a PicoTipTM (New Objective, Woburn, MA) fitted on a Z-spray (Micromass) interface. Chromatographic separations were performed on a reversed-phase capillary column (Pepmap C18, 75 µm i.d., 15 cm length; LC Packings) with a flow rate of 200 nl/min. The chromatography was carried out using a linear gradient from 5 to 50% solvent B in 1 h and from 5 to 80% solvent B in 1.5 h (solvent A: H2O/formic acid (FA), 99.9/0.1, v/v; solvent B: H2O/acetonitrile/FA, 19.92/80/0.08, v/v/v). Eluting peptides were detected at a wavelength of 210 nm prior to analysis by electrospray ionization (ESI)-MS/MS in a quadrupole time-of-flight (Q-TOF) mass spectrometer using a nanoelectrospray ion source. Data acquisition was controlled by MasslynxTM software (Micromass) using a manual acquisition mode for MS and MS/MS experiments. Typically, the capillary voltage was set to 1800 V and the counter electrode was set to 40 V. Low-energy collision-induced dissociation (CID) was performed using argon as a collision gas (pressure in the collision cell was set to 3–4 x 10–5 mbar), and the collision energy was optimized manually for all precursor ions (in the range of 25–35 eV).

High-pressure Liquid Chromatography (HPLC) Separation of CHH and MIH from SG Extract—
Separation of extracts from 30 SG of O. limosus was performed on a HPLC system consisting of two model 510 pumps, a model 680 gradient controller, a model 481 ultraviolet (UV) detector set at 210 nm (all Waters, Eschborn, Germany) and a data acquisition system (Bischoff Chromatography, version 1.52 f, Leonberg, Germany). The chromatography was carried out on a Luna® 5 µ-phenyl-hexyl column (25 x 0.46 cm; Phenomenex, Aschaffenburg, Germany) with a linear gradient from 50 to 80% solvent B in 1 h at a flow rate of 0.9 ml/min. Solvent A was composed of 0.11% TFA; solvent B was composed of 0.10% TFA and 60% acetonitrile. CHH and MIH peaks were collected manually, immediately dried, and stored under nitrogen at –20 °C.

NanoESI-QTOF MS of CHH and MIH—
For nanoESI-QTOF MS, the dried HPLC fractions of CHH and MIH were dissolved in 5 µl of distilled water. The samples were diluted 1/5 in methanol/water/acetic acid (50/25/25, v/v/v) prior to mass spectrometry. The capillary voltage was set to 1100 V and the counter electrode was set to 40 V.


    RESULTS
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Direct identification of neuropeptides was achieved by analysis of the desalted tissue extract obtained from the neurosecretory XO-SG of O. limosus. The purification procedure on Sep-Pak C18 permitted the selective enrichment of small to medium polypeptides, as visible from the UV trace (Fig. 1). A clear-cut chromatogram showing a relatively low number of predominant peaks can be obtained only if excised SGs are immediately frozen in liquid nitrogen and rapidly extracted. The most abundant polypeptides were identified as CHH precursor-related peptides A/A* (Fig. 1, LC fractions 3 and 4), which are associated with the CHH of O. limosus (12, 13). They are characterized by their 4-fold charged molecular ions at m/z 886.63 and 879.88, corresponding to two 33-amino acid-containing species, respectively (Table I). By selecting the precursor ions of these already known CPRPs, an amino acid sequence coverage of 76% was achieved using the tandem MS fragmentation by low-energy QTOF CID. In Fig. 2, the part of the fragmentation spectrum of Orl-CPRPA* showing the serine-rich region 13–20 is depicted.



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FIG. 1. NanoLC-separation UV profile of the peptide extract equivalent to 0.5 SG of O. limosus. The m/z-values of 11 components, obtained by nanoLC-ESI-MS, are listed in the inset along with their corresponding LC fraction number. Sequence data thus obtained for fractions 1–11 by low-energy CID are summarized in Table I. Chromatographic conditions were as follows: Pepmap C18 column (75 µm i.d., 15 cm length; LC Packings), elution with a linear gradient from 5 to 50% solvent B in 1 h at a flow rate of 200 nl/min. Solvent A: 0.1% FA; solvent B: 0.08% FA, 80% acetonitrile.

 

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TABLE I Peptides identified from the SG extract of O. limosus

Table 1 depicts the amino acid sequences obtained by use of low-energy CID of all LC fractions investigated (CPRPA/A*, CHH precursor related peptide A/A* of O. limosus (12, 13), PDH, pigment-dispersing hormone of O. limosus (14)). LC fractions 1, 2, 5, and 6 were identified as CPRPA/A* variants (v). ND, not detected. X, amino acids not identified by low-energy CID fragmentation.

 


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FIG. 2. Part of the low-energy QTOF CID spectrum and amino acid sequence of the quadruply charged molecular ion of CPRPA* at m/z 886.63 (LC fraction 3) from O. limosus. Identified amino acids are in bold. Amino acid sequence coverage of 76% was achieved for both known CPRPs. The m/z values of the y-ions obtained are listed in the inset

 
Additionally, four variants of CPRPs, corresponding to LC fractions 1, 2, 5, and 6 (Fig. 1), were detected as 4-fold charged molecular ions by nanoLC-MS at m/z 890.62, 883.90, 877.37, and 848.89, respectively (Table I, spectra not shown). Their sequence coverage obtained by this approach of ~64% achieved for all variants investigated was not sufficient for the determination of all amino acid substitutions and/or modifications. The sequence data are summarized in Table I. By tandem mass spectrometric investigation of CPRP-variants v1 and v2 (LC fractions 1, 2), identical MS/MS sequence spectra (data not shown) with respect to the structurally characterized CPRPs A/A* were obtained. The observed mass difference of 16 Da between CPRPv1/CPRPA* and CPRPv2/CPRPA, respectively, might be interpreted as a single amino acid substitution or as a modification, like oxidation of the methionine in position 9 of CPRPs A/A*. In contrast to this, one amino acid substitution in position 30, histidine instead of glutamine, could be assigned unambiguously to the CPRP variants 3 and 4, as correlated to LC fractions 5 and 6 (data not shown), indicating the existence of a new peptide variant of CPRP in the SG of O. limosus. Nevertheless, the observed mass differences for all CPRP variants may be caused in general by several amino acid substitutions in the region in which no fragment ions were obtained. Full characterization of these four CPRP variants remains still to be established. Under the standard conditions used for the on-line LC QTOF-low-energy CID of the LC fractions 7 and 8, no fragment ions were observed. LC fraction 9 was identified by on-line LC-MS/MS as the already known PDH of O. limosus (14), whereas in LC fractions 10 and 11 two new genetic variants of this hormone, PDHB and PDHC (Swiss-Prot accession nos. P83586 and P83587, respectively), were detected. A complete sequence coverage of both new PDH variants beside the already known one could be obtained by LC-MS/MS. The low-energy CID spectra and their corresponding amino acid sequences are depicted in Fig. 3. These new PDH forms contain an unusual structural feature with respect to the C-terminal amino acid, not described before. All crustacean PDH peptides described up to now exhibited an amidated alanine (A-NH2) as C-terminal residue (11). In contrast to this, the new genetic variants PDHB and PDHC carry an amidated valine (V-NH2) as C-terminal amino acid. The biological significance of this structural feature remains to be clarified.



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FIG. 3. Low-energy QTOF CID mass spectra and resulting amino acid sequences of the doubly protonated quasi molecular ions of PDH, PDHB, and PDHC at m/z 970.97, 936.92, and 928.95, respectively (corresponding to LC fractions 9, 10, and 11). Identical amino acids are in bold. The fragment ions obtained are summarized in Table II.

 
If SG peptide extract has been separated by a gradient up to 80% solvent B, we were able to detect it in the UV profile peptides, which were potential CHH (12) and MIH (Swiss-Prot accession no. P83636) candidates (Fig. 4). However, by monitoring the total ion current, we could prove that no ions from the large peptide components like CHH and MIH were formed under the standard conditions used in our experiments. To inspect this, SG extract has been exposed to a preparative HPLC (data not shown). Fractions, which were potential CHH and MIH candidates, were submitted separately to the nanoESI-MS under different, lower pH conditions, under which the purified large peptide components were well ionized and identified according to their multicharged molecular ions (Fig. 5).



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FIG. 4. NanoLC-separation UV profile of the peptide extract equivalent to 0.5 SG of O. limosus. Identified neuropeptides obtained by nanoLC-ESI-MS are listed in the inset along with their corresponding LC fraction number. ?, not identified components. Chromatographic conditions were modified as follows: elution with a linear gradient from 5 to 80% solvent B in 1.5 h.

 


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FIG. 5. NanoESI-QTOF mass spectra of CHH (A) and MIH (B) obtained from HPLC-separated SG of O. limosus. The spectra were recorded in the positive ion mode using methanol/water/acetic acid (50/25/25, v/v/v) as solvent.

 

    DISCUSSION
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
This is the first report on the direct characterization of neuropeptides from the crustacean XO-SG neurosecretory system by nanoLC-MS. The precision, reproducibility, and accuracy of LC-MS for the analysis of polypeptides is well documented (1923). SG peptides from O. limosus were isolated from organic and inorganic salts by reversed-phase Sep-Pak C 18, prior to nanoLC-MS. Several peptide hormones of the SG could be identified and sequenced by nanoLC-MS/MS. The SG peptides CPRP A/A* (12, 13) and PDH (14) from O. limosus could be identified (CPRP A/A*) or fully characterized (PDH) by a sequence coverage of 100% (Tables I and II). Additionally, the existence of four CPRP variants (CPRP v1–4) and two new genetic PDH variants (PDHA and PDHB) not observed using conventional chromatographic systems (12, 13) or molecular cloning (14, 15) was proved.


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TABLE II m/z values of fragment ions and resulting sequence data of PDH, PDHB, and PDHC obtained by low-energy QTOF CID

Identical amino acids are in bold.

 
Large peptides belonging to the CHH and MIH family could not be detected under the standard conditions used in our experiments. These peptides were submitted separately to the nanoESI-MS under different, lower pH conditions, under which these purified large components were well ionized and identified according to their multicharged molecular ions. These results are in agreement with previous observations (24). We presume that the tertiary structure of these native peptides containing disulfide bridges can largely influence the ionization process in the electrospray ion source. The described conditions for the nanoESI-MS experiment, however, were not compatible with our nanoLC column system. Alternatively, in an off-line approach, fractions obtained by a nanoLC separation could be subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for determination of molecular ions. A potential of MALDI-TOF/TOF MS for sequencing of native polypeptides, however, has not been explored yet.

The question, if other peptides like RPCH, PDH precursor-related peptide (PPRP), Phe-Met-Arg-Phe-NH2 (FMRFamide)-like peptides, and enkephalins should be detectable in our peptide mixture from a SG system is not clear at this moment.

Within the study for structural characterization of chromatophorotropic neuropeptides in the SG of the prawn Penaeus japonicus, RPCH has been preparatively purified and identified by Edman sequencing as a component, but the amount of the animal gland tissue material used in this study, however, was 24,000 times higher than in our study for the on-line LC/MS identification (11). Even using this relatively large amount of material, the PPRP, located between the signal peptide and the native PDH hormone (14), was not described to be present as a component of the SG. The question of whether the PPRP is transported and stored in the SG system has not been clarified yet (14).

In the case of FMRFamide-like peptides of the prawn Penaeus monodon, which were found to be components of the SG, the amount of material for purification was 18,000 times higher than in our study (25). On the other hand, enkephalins were postulated to be present in SG of crustacean by cytochemical methods (26), but never found by structural analysis. In the same context, it can be hypothesized that either the sensitivity of our system did not allow detection of attomolar amounts of these components or that they were not present at all. Additionally, a number of very low abundant components visible in the LC UV trace were detected as singly charged molecular ions. Low-energy CID MS/MS experiments on these low molecular mass compounds (Mr < 600 Da) did not reveal any structural information.

The results obtained in this study demonstrate the high potential of the nanoLC-MS techniques with respect to identification and sequencing of peptides from biological sources without prior purification steps. Another benefit of this technique is the high sensitivity, because only 0.5 SG equivalents were required for the full characterization of PDH peptides. Quantification of SG peptides for analysis of specific and already characterized components has been generally performed by immunochemical methods (16, 17), limiting this approach by availability of specific antibodies to only well-characterized peptides. In addition, the problem of antibody cross-reactivity is also avoided by use of direct mass spectrometric methods. In perspective, nanoLC-MS allows the quantification and de novo identification/sequencing of peptides if a calibration with the analyte is applied.

In recent studies for characterization of peptides from neurosecretory systems, strategies to restrict a number of components for analysis either by a preseparation step prior to the on-line nanoLC-MS experiment or by a limitation of the mass range detection were applied. For analysis of peptides from the central nervous system of Drosophila larvae, where 50 brain organ equivalents were used, the extract was preseparated by size-exclusion chromatography (27). In another study, for identification of peptides in Sepia officinalis, a high-flow LC/ESI-ion trap-MS/MS was used under restricted mass detection of 600 Da (28).

In the here-described approach, identification of already known and of previously not characterized, novel peptide hormone structures in neurosecretory systems can be accomplished. The limits of the method by restricted ionizability of peptide components can be overcome by comparison of the LC-UV trace and the total ion current in the MS analyzer. For detection of very minor components in complex peptide/protein mixtures, which may be relevant in the full-proteome expression analysis of glands, a higher amount of tissue must be prepared and subjected to an array of multidimensional separation techniques, such as multidimensional LC, capillary electrophoresis, or two-dimensional gel electrophoresis, and be combined with advanced and highly sensitive mass spectrometric methods.


    FOOTNOTES
 
Received, August 12, 2003

Published, February 20, 2004

Published, MCP Papers in Press, February 22, 2004, DOI 10.1074/mcp.M300076-MCP200

1 The abbreviations used are: XO-SG, X-Organ sinus gland; CHH, crustacean hyperglycemic hormone; CID, collision-induced-dissociation; CPRP, CHH precursor-related peptide; ESI, electrospray ionization; QTOF, quadruple time-of-flight; MS, mass spectrometry; FA, formic acid; nanoLC-MS, nanoscale liquid chromatography mass spectrometry; PDH, pigment-dispersing hormone; SG, sinus gland; TFA, trifluoroacetic acid; MIH, moult-inhibiting hormone; VIH, vitellogenesis-inhibiting hormone; GIH, gonad-inhibiting hormone; RPCH, red pigment-concentrating hormone; HPLC, high-pressure liquid chromatography; UV, ultraviolet; PPRP, PDH precursor-related peptide. Back

* This work was supported by the Deutsche Forschungsgemeinschaft through grants to J. P.-K. (PE 415/15-1,2) and R. K. (Ke 206/17-1,2). The QTOF instrument was purchased from a HbfG Grant (Land Nordrhein Westfalen) to J. P.-K. The article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Institute for Medical Physics and Biophysics, Robert-Koch Straße 31, University of Münster, D-48149 Münster, Germany. Tel.: 49-251-83-52308; Fax: 49-251-83-55140; E-mail address: jkp{at}uni-muenster.de


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 DISCUSSION
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