Peptidomics-Based Discovery of Novel Neuropeptides Marcus Svensson,†,⊥ Karl Sko1 ld,†,⊥ Per Svenningsson,‡ and Per E. Andren*,†,§ Biological and Medical Mass Spectrometry Research Laboratory, Department of Pharmaceutical Biosciences, Uppsala University, SE-75124 Uppsala, Sweden, Department of Physiology and Pharmacology, Karolinska Institutet, SE-17177 Stockholm, Sweden, and Amersham Biosciences, Bjo¨rkgatan 30, SE-75184 Uppsala, Sweden Received October 15, 2002
Modern proteomic methodologies have significantly improved the possibilities of large-scale identification of proteins. However, these methodologies are limited by their inability to reliably detect endogenously expressed peptides. We describe a novel approach of combining sample preparation, comprising focused microwave irradiation and mass spectrometric peptide profiling that has enabled us to simultaneously detect more than 550 endogenous neuropeptides in 1 mg of hypothalamic extracts. Automatic switching tandem mass spectrometry and amino acid sequence determination of the peptides showed that they consist of both novel and previously described neuropeptides. The methodology includes virtual visualization of the peptides as two- and three-dimensional image maps. In addition, several novel and known post-translational modifications of the neuropeptides were identified. The peptidomic approach proved to be a powerful method for investigating endogenous peptides and their post-translational modifications in complex tissues such as the brain. It is anticipated that this approach will complement proteomic methods in the future. Keywords: brain • electrospray • hypothalamus • mass spectrometry • nanoLC • neuropeptides • peptidomics • peptide processing • post-translational modifications
Introduction Peptides exert potent biological actions in the respiratory, cardiovascular, endocrine, inflammatory, and nervous systems. In the central nervous system, most neurons contain biologically active peptides together with conventional neurotransmitters.1 Neuropeptides are implicated in the pathology of various neurological and psychiatric disorders such as depression, Parkinson’s disease, and eating and sleep disorders.2 Most neuropeptides are synthesized as propeptide precursors that are processed and activated via limited proteolysis by a number of specific convertases. The sites of proteolysis are generally at pairs of basic amino acids or, less frequently, at single basic residues in concordance with prohormone convertase activity.3,4 These processed bioactivated peptides are secreted into the extracellular space. During this process, the peptides may undergo functionally important post-translational modifications, e.g., glycosylation, C-terminal amidation, acetylation, phosphorylation, and sulfation.2 Neuropeptides exert their actions by acting on specific receptors localized on target cell surfaces and thereby modulating various intracellular signal transduction pathways.2 In the past, properties predominantly possessed by biologically active peptides have been successfully used for isolation of putative novel neuropeptides.4-7 The sequences of these * To whom correspondence should be addressed. Phone: +46-18 6120136. Fax: +46-18 6121826. E-mail:
[email protected]. † Biological and Medical Mass Spectrometry Research Laboratory, Department of Pharmaceutical Biosciences, Uppsala University. ‡ Department of Physiology and Pharmacology, Karolinska Institutet. § Amersham Biosciences. ⊥ Equal contribution to this work. 10.1021/pr020010u CCC: $25.00
2003 American Chemical Society
peptides have in most cases been determined by extensive purification steps followed by Edman degradation. Evidently, this strategy has led to the discovery of numerous biologically important peptides2,8 but its low sensitivity and requirement of high sample amount is a considerable drawback. Recently, mass spectrometry (MS) has been used to make advances in the area of direct analysis of biological molecules due to its high sensitivity and the possibility to combine it with aqueousbased buffer systems. Capillary nanoscale liquid chromatographic (nanoLC) separation coupled on-line to electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF) MS is uniquely advantageous for the study of complex native neuropeptide mixtures.9 Additional analysis by tandem MS (MS/MS) using collision-induced dissociation (CID) fragmentation reveal the amino acid sequences and thus the identity of the selected peptides. However, experiments using MS for neuropeptide profiling in tissues have previously been hampered by the complex and time-consuming purification steps.10,11 In the present study, we have developed a novel approach to study a large number of neuropeptides and employed it to an investigation of the endogenous neuropeptide content of hypothalamic brain tissue samples from rats and mice. Using this novel combination of sample preparation protocol and nanoLC-ESI-Q-TOF-MS, we were able to detect more than 550 endogenous peptides. Several of these neuropeptides were novel, whereas others were previously described. Moreover, unknown post-translational modifications of some of these peptides were also identified. Thus, this methodological approach makes it possible to extensively investigate the mammalian brain peptidome. Journal of Proteome Research 2003, 2, 213-219
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Experimental Section Chemicals. Glacial acetic acid (100%) of analytical grade was obtained from Merck (Darmstadt, Germany). Acetonitrile (HPLC grade) and poly(ethylene glycol) (PEG) 200 were obtained from Merck (Darmstadt, Germany), poly(ethylene glycol) 400 and 600 were purchased from Sigma (St. Louis, Missouri). Water from a Milli-Q Gradient A10 System was used (Millipore, Bedford, Massachusetts). The deuterated neuropeptides substance P 1-7, substance P 1-11, met-enkephalin, and neurotensin were synthesized by Dr. G. Lindeberg at the Department of Medicinal Chemistry, Division of Organic Pharmaceutical Chemistry, Uppsala University, Uppsala, Sweden. Sample Preparation. Rats (Sprague-Dawley) or mice (C57/ Bl6) were sacrificed by focused microwave irradiation (4.5-5 kW for 1.4 s) using a small animal microwave (Murimachi Kikai, Tokyo, Japan). Hypothalamus was thereafter rapidly dissected out and stored at -80 °C. The brain tissue was suspended in cold extraction solution (0.25% acetic acid) and homogenized by microtip sonication (Vibra cell 750, Sonics & Materials Inc., Newtown, Connecticut) to a concentration of 0.2 mg tissue/ µL. The suspension was centrifuged at 20 000 × g for 30 min at 4 °C. The protein- and peptide-containing supernatant was transferred to a centrifugal filter device (Microcon YM-10, Millipore, Bedford, Massachusetts) with a nominal molecular weight limit of 10 000 Da, and centrifuged at 14 000 × g for 45 min at 4 °C. Finally, the peptide filtrate was immediately frozen and stored at -80 °C until analysis. Nanoliter Flow Liquid Chromatography Mass Spectrometry. Five µL peptide filtrate (equivalent to 1.0 mg hypothalamic tissue) was injected onto a fused silica capillary column (75 µm i.d., 15 cm length), packed with 3 µm diameter reversed phase C18 particles (NAN75-15-03-C18PM, LC Packings, Amsterdam, The Netherlands). The particle bound sample was desalted by an isocratic flow of buffer A (0.25% acetic acid in water) for 35 min and eluted during a 60 min gradient from buffer A to B (35% acetonitrile in 0.25% acetic acid), which was delivered using an Ultimate LC system (LC Packings, Amsterdam, The Netherlands). The eluate was directly infused into the ESI-Q-TOF mass spectrometer (Q-Tof, Micromass Ltd., Manchester, United Kingdom) at a flow rate of 120 nL/min for analysis.9 The in-house manufactured spray emitter was manually drawn from a 75 µm i.d., 220 µm o.d. fused silica capillary (Polymicro Technologies Inc., Phoenix, Arizona,) to obtain a tapered spray tip with an i.d. of approximately 5 µm and an o.d. of 10 µm. The electrospray potential of 1.9 kV was applied to a zero dead volume metal union (Valco Instruments Co. Inc., Houston, Texas) placed 5 cm from the emitter tip. Data acquisition from the ESI-Q-TOF instrument was performed in continuous mode and mass spectra were collected at a frequency of 3.6 GHz and integrated into a single spectrum each second. The time between each such spectrum was 0.1 s. The parameter settings were as follows: Cone 39 V, extractor 3 V, RF lens 1.49, source temperature 80 °C, focus 0 V, ion energy 1.8 eV, collision energy 10 eV, and multiple channel plate detector (MCP) 2100 V. The cone gas flow rate was set to about 100 L/h. In the wide band-pass quadrupole mode of the mass spectrometer, mass spectra were collected in the mass-to-charge (m/z)-ratio range of 300-1000 with a mass resolution of 6400 (fwhm) at m/z 558.31. Mass calibration was made from an infusion of a PEG 200, 400, and 600 mixture, (25, 50, and 75 ng/mL, respectively) in 2 mM ammonium 214
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Figure 1. Two-dimensional graph of the neuropeptide content from one mg of rat hypothalamus. The neuropeptide map displays peptides in the m/z range 300-1000 in 60-min gradient elution from the nanoLC separation. Spot intensity is represented by color changes, with black being the most intense reading and white the lowest.
acetate, 50% methanol and 0.2% formic acid, according to the recommendations of the manufacturer. Mass spectrometry data collected during the 60-min chromatographic peptide separation was exported as a text file using MassLynx DataBridge. The mass information was prepared using a proprietary software tool currently being developed (Amersham Biosciences, Uppsala, Sweden) to visualize the elution profile both as a virtual 2-D gel and as a 3-D graph. A sensitivity assessment of the experimental system was performed by injections of a mixture of deuterated peptide standards (500 amol, 1 fmol, or 5 fmol in 5 µL) to the nanoLCESI-Q-TOF-MS system. The synthetic peptides were concentrated, separated and analyzed as described above. Peptide Characterization and Identification. Sequence information of the peptides was obtained from precursor ions (peptides) by an automatic switching function of the Q-TOF software from MS to MS/MS mode. The precursor ions were automatically selected for fragmentation during four nanoLC separations and subsequently put in an exclusion list for 200 s. The switching was intensity dependent with the threshold value set to 12 ion counts. The collision chamber was filled with argon with the inlet pressure set to about 15 psi. The collision energy was ramped from 23 to 31 eV in 5 s. The collected collision-induced dissociation fragmentation spectra were integrated into a single spectrum twice every second in the m/z-ratio range of 40-1200. These spectra were deconvoluted using MaxEnt3 (MassLynx 3.4, Micromass Ltd.) and interpreted by the BioLynx (MassLynx 3.4) software tools and/or manually. The proposed peptide sequences were compared with the nonredundant database of National Center for Biotechnology Information (NCBI) to establish the peptide identities using Basic Local Alignment Search Tool (BLAST) “search for short nearly exact matches” (http://www. ncbi.nlm.nih.gov/BLAST).
Results More than 800 peptide ions producing distinct MS peaks (corresponding to 550 endogenous neuropeptides) were detected during the 60-min nanoLC-ESI Q-TOF-MS analysis of hypothalamic brain tissue from rats and mice. The MS data was processed using a proprietary computer program and
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Novel Hypothalamic Neuropeptides
Figure 2. Selected region of a neuropeptide map of the rat hypothalamus displayed as two- and three-dimensional graphs. Both graphs present the nanoLC elution profile for 19.2-29.6 min and the m/z range 548.7-577.8. a, The distance between the coeluting parallel lines correspond to the charge states of the ions. Relative spot intensity in the two-dimensional graph is represented by color changes, black being the most intense reading and white the lowest. The seemingly unresolved spots at 20.9 min, m/z 552.2 [M + 9H]9+ and 22.6 min, m/z 549.2 [M + 8H]8+ are in fact resolved by further zooming. b, Three-dimensional graph showing the relative intensity of identified secretogranin I peptide (m/z 549.85 [M + 2H]2+), neurokinin A (m/z 567.35 [M + 2H]2+), neurotensin (m/z 558.31 [M + 3H]3+), met-enkephalin (m/z 574.29 [M + H]+), melanotropin alpha (m/z 569.66 [M + 3H]3+), leu-enkephalin (m/z 556.35 [M + H]+). Table 1. Novel Peptides Identified from Hypothalamic Tissue of Rat and Mouse Using Capillary Nanoflow Liquid Chromatography and Electrospray Quadrupole Time-of-Flight Tandem Mass Spectrometry precursora
chromogranin A precursor cocaine-amphetamine-regulated transcript protein (CART) cocaine-amphetamine-regulated transcript protein (CART) neurosecretory VGF protein neurosecretory VGF protein pituitary adenylate cyclase activating polypeptide (PACAP) proenkephalin A precursor proenkephalin A precursor proenkephalin A precursor proenkephalin A percursor proenkephalin A, B, pro-opiomelanocortin (POMC) proenkephalin B precursor prohormone convertase 2 pro-MCH precursor pro-opiomelanocortin (POMC) secretogranin I precursor secretogranin II precursor stathmin vasopressin-neurophysin 2-copeptin
acc no: rat/mouseb
sequencec
speciesd
P10354/P26339 P49192
395AYGFRDPGPQL405/392AYGFRDPGPQL402 82IPIYE86
rat/mouse rat
P49192
rat rat/mouse rat/mouse
P13589
37ALDIYSAVDDASHEKELPR55 Ser48 phosphorylation 491PPEPVPPPRAAPAPTHV507 180pyroglutamic acid QETAAAETETRTHTLTRVNLESPGPERVW209 111GMGENLAAAAVDDRAPLT128
P04094/P22005 P04094 P04094 P04094/P22005 P04094, P06300, P01194
198SPQLEDEAKELQ209 198SPQLEDEAKEL208 264GGFMRF269 219VGRPEWWMDYQ229 YGGF multivalent
rat/mouse rat rat rat/mouse rat
GI:204040 P28841/P21661 P14200 P01193 O35314 Q03517 P13668 P01186
133SSEMAGDEDRGQDGDQVGHEDLY155 94IKMALQQEGFD104 131EIGDEENSAKFPIG144 acetylation-205YGGFMTSEKSQTPLVTL221 585SFAKAPHLDL594 300ESKDQLSEDASKVITYL316 acetylation-2ASSDIQVKELEKRASGQAF20 151VQLAGTQESVDSAKPRVY168
rat rat/mouse rat mouse rat mouse rat rat
P20156/P20156/-
rat
a Peptide precursor name. b Precursor accession number in SWISS-PROT or NCBI. c Amino acid sequence of the identified peptide and localization in the precursor. d Species in which the peptide has been identified.
displayed as two- or three-dimensional virtual peptide maps (Figures 1 and 2). In this way comprehensive information about the peptides is presented, i.e., elution time, mass-to-charge ratio, charge state and relative intensity which improve the visualization for qualitative and semiquantitative interpretation (Figure 2). The produced peptide profile maps consisted of both novel and known neuropeptides that were sequenced and identified using MS/MS analysis. Peptide Identification and Characterization. The Q-TOF mass spectrometer was set to an automatic function-switching mode (MS-MS/MS-MS) during analysis for sequence identi-
fication of the endogenous hypothalamic peptides. The MS software selected approximately 10% of the precursor ions from each sample injection for fragmentation using CID during the 60-min gradient. The generated MS/MS spectra were interpreted manually and/or by software from the N-terminal (bions) and C-terminal (y-ions) producing the amino acid sequences (Figure 3). Matching of the sequences from the MS/ MS spectra using protein databases identified neuropeptides with masses ranging up to 4960 Da (Tables 1 and 2). Novel Neuropeptides. Several neuropeptides that have not previously been described in the literature were identified. Journal of Proteome Research • Vol. 2, No. 2, 2003 215
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Figure 3. Tandem mass spectra of selected neuropeptides. a, b, The sequences of the C-terminally amidated joining peptide derived from pro-opiomelanocortin precursor in rat and mouse, respectively. c, The complete b- and y-ion series of peptide I 1-12, which is derived from the proenkephalin A precursor, and d, the sequence identification of the neuropeptide lipotropin gamma. The MS/MS fragment ion labels used are based on the Roepstorff nomenclature.27
These originated from known neuropeptide-containing precursors and one novel neuropeptide originated from stathmin, a protein not known to be a peptide precursor (Table 1). The majority of the identified novel peptides were proteolytically cleaved at pairs of the basic amino acids lysine and arginine, or less frequently, at single basic residues. However, two neuropeptides originating from neurosecretory protein VGF and the protein stathmin were processed at nonbasic sites. The proteolytic cleavage sites of the novel peptides in the proenkephalin A, chromogranin A, neuroendocrine protein VGF, and pro-opiomelanocortin precursors were located at the same relative positions in both the rat and mouse. The proopiomelanocortin precursor-derived joining peptide was posttranslationally amidated and contained multiple inter-species amino acid substitutions (Figure 3a and b). In addition, we identified two forms of the peptide I (1-11 and 1-12), originating from the proenkephalin A precursor (Figure 3c). 216
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Known Neuropeptides. Many of the classical neuropeptides, such as neurotensin, substance P, neurokinin A, corticotrophinlike intermediate lobe peptide (CLIP), and beta-endorphin were identified both in rat and mouse brain tissue (Table 2). The two most abundant MS peaks during the 60-min elution were sequenced and identified as the 4.96 and 4.93 kDa thymosin beta-4 and thymosin beta-10 peptides, respectively. From the proSAAS precursor, big PEN and little SAAS were identified. Moreover, relatively high levels of the enkephalin variants leucine-enkephalin (leu-enk), methionine-enkephalin (metenk), met-enkRF, met-enkRGL/met-enkRSL (rat/mouse), as well as the 4.44 kDa lipotropin gamma peptide from the proopiomelanocortin precursor were sequenced and identified (Figure 3d). Furthermore, CLIP was identified containing Arg in the N-terminal. Post-translational Modifications of Neuropeptides. A novel peptide from the cocaine- and amphetamine-regulated tran-
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Table 2. Known Endogenous Peptides Identified from Hypothalamic Tissue of Rat and Mouse Using Capillary Nanoflow Liquid Chromatography and Electrospray Quadrupole Time-of-Flight Tandem Mass Spectrometry precursora
cerebellin chromgranin A precursor neurotensin/neuromedin N proenk A precursor, POMC
peptideb
acc no: rat/mousec
proenkephalin A precursor proenkephalin A precursor progonadoliberin 1 pro-MCH precursor pro-opiomelanocortin
P23436 P26339 P20068/Q9D3P9 P04094, P01194/ P22005, P01193 leu-enk P04094, P06300/ P22005, P06300 met-enkRGL/met-enkRSL P04094/P22005 met-enk RF P04094/P22005 progonadoliberin I P07490 neuropeptide E-I P14200 melanotropin alpha P01194/P01193
pro-opiomelanocortin pro-opiomelanocortin
CLIP lipotropin gamma
pro-opiomelanocortin
beta-endorphin
pro-opiomelanocortin
joining peptide
proSAAS
big PEN
proSAAS prosomatostatin protachykinin 1 protachykinin 1 protachykinin 1 thymosin beta-4
little SAAS somatostatin-28 (1-12) neurokinin A substance P C-term flanking peptide thymosin beta-4 short form thymosin beta-10
proenk A, proenk B
thymosin beta-10
cerebellin WE-14 neurotensin met-enk
sequenced
speciese
1SGSAKVAFSAIRSTNH16 358WSRMDQLAKELTAE371 150pyroglutamic acid LYENKPRRPYIL162 YGGFM, multivalent
rat mouse rat/mouse rat/mouse
YGGFL, multivalent
rat/mouse
188YGGFMRGL195/188YGGFMRSL195 263YGGFMRF269 24pyroglutamic acid HWSYGLRPG33-amidation 131EIGDEENSAKFPI143-amidation 124SYSMEHFRWGKPV136-amidation Ser124, Ser126 acetylation P01193 141RPVKVYPNVAENESAEAFPLEF162 P01193 165ELEGERPLGLEQVLESDAEKDDGPYRVEHFRWSNPPKD202 P01193 acetylation-205YGGFMTSEKSQTPLVTLFKNAIIKNAH231 P01194/P01193 103AEEETAGGDGRPEPSPRE120-amidation/ 103AEEEAVWGDGSPEPSPRE120-amidation Q9QXU9/Q9QXV0 243LENSSPQAPARRLLPP258/ 245LENPSPQAPARRLLPP260 Q9QXU9/Q9QXV0 42SLSAASAPLAETSTPLRL59 P01167 89SANSNPAMAPRE100 P06767/P41539 98HKTDSFVGLM107-amidation P06767/P41539 58RPKPQQFFGLM68-amidation P06767 111ALNSVAYERSAMQNYE126 P01253/P20065 acetylation-2SDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES44 -/GI:13384628 acetylation-2ADKPDMGEIASFDKAKLKKTETQEKNTLPTKETIEQEKRSEIS44
rat/mouse rat/mouse rat rat rat/mouse mouse mouse mouse rat/mouse rat/mouse rat/mouse rat rat rat/mouse rat/mouse rat/mouse rat/mouse
a Peptide precursor name. b The name of the peptide. c Precursor accession number in SWISS-PROT or NCBI. d Amino acid sequence of the identified peptide and localization in the precursor. e Species in which the peptide has been identified.
script (CART) protein was identified in the rat hypothalamus carrying a phosphorylation at Ser48. The neuropeptide CLIP was sequenced and identified in mouse with and without a phosphate group at Ser154. Melanotropin alpha, which carries a C-terminal amidation in rat and mouse, was found to contain two additional modification sites and states. Ser124 and Ser136 were acetylated in both species and in the mouse a variant lacking the Ser136 acetylation was also identified. A neuropeptide E-I variant originating from the pro-MCH precursor protein was identified lacking its C-terminal isoleucine amide. Two forms of the relatively abundant thymosin beta-4 were discovered of which the less abundant form was oxidized at the Met7 position. One of the novel peptides from the neuroendocrine protein VGF had a pyroglutamic acid modification of the Gln180 residue.
Discussion In the present report, we describe a methodological approach to detect and identify a large number of endogenous neuropeptides in complex tissue extracts from the hypothalamus of rats and mice. There are several major findings of this study. First, many of the identified peptides represent previously uncharacterized and novel processed fragments of protein precursors. Second, a novel peptide was discovered from a protein previously not known to be a neuropeptide precursor. Third, novel post-translational modifications of neuropeptides were identified and located at specific amino acids in both novel and previously characterized neuropeptides.
Peptides are present in all parts of the nervous system and each peptide has its own unique distribution pattern. The hypothalamus is a phylogenetically ancient region of the mammalian brain mediating processes such as reproduction, lactation, fluid balance, and metabolism. Moreover, the hypothalamus moderates aspects of behavior, such as circadian rhythms, basic emotions, feeding and drinking, mating activities, and response to stress, as well as normal development of the immune system.2 As briefly mentioned in the Introduction, these processes involve neuropeptides. Thus, the hypothalamus is an ideal source for studies of functionally relevant neuropeptides and their post-translational modifications. The methodological novelty in this report is the combination of sample preparation comprising focused microwave irradiation and MS peptide profiling. To avoid protein degradation and to obtain an unaltered concentration of the neuropeptides, the rats and mice were sacrificed by focused microwave irradiation. Such irradiation raises the brain temperature to 90 °C within 1.4 s and thereby rapidly prevents enzymatic degradation.12-14 Post mortem activity of proteases has been shown to play an important role on the level of the peptide concentrations in the brain,13,15 as well as for detecting posttranslational modifications of proteins and peptides.16,17 In previous experiments using traditional (nonmicrowave irradiation) sacrificing methods, fragments from hemoglobin were a major peptide source detected at high levels in the brain.9,11,18-20 Interestingly, none of the peptides detected in the present study were identified as hemoglobin fragments. Furthermore, in our Journal of Proteome Research • Vol. 2, No. 2, 2003 217
research articles previous investigation of hypothalamic tissue from decapitated rats we detected a C-terminally degraded form of thymosin beta-4, which was not detected in the present study. These findings suggest that substantial protein/peptide degradation occurs in hypothalamic tissue within minutes post mortem.9 The microwave irradiation procedure of the brain tissue therefore has several advantages; it enables the relatively lowabundant neuropeptides to remain intact, it minimizes degradation of proteins by proteolysis, and it conserves the posttranslational modifications of the neuropeptides. The present study showed for the first time that it was possible to detect and identify a large number of previously characterized and novel neuropeptides from a single sample using only one mg of brain tissue (Figure 1, Tables 1 and 2). One of the novel peptides found in this study originated from the protein prohormone convertase (PC2), which is involved in pro-hormone and pro-neuropeptide processing.21 The activation and regulation of PC2 is not fully understood, but the novel peptide may provide important information: shortly after its biosynthesis, pro-PC2 specifically associates with a small acidic protein named pro-7B2 within the endoplasmatic reticulum. Both pro-7B2 and pro-PC2 are cleaved by furin, yielding an extended form of the identified peptide in the present study and a 7B2 peptide. However, these peptide products remain attached to the active site of PC2 zymogen, thus maintaining PC2 in a maturation-conducive conformation. The PC2 self-activates by intramolecular cleavage, releasing the novel peptide identified in the present study and allowing the PC2 convertase to be active. The PC2-derived peptide identified from the PC2 prodomain may play an important role in the regulation of the activity of the PC2 convertase. Recently, a novel peptide was discovered in in vivo microdialysis samples,22 which was identified as the N-terminal cleavage product of peptide I23 (peptide I 1-10), and processed from the pro-enkephalin A precursor. We have identified a processing variant, peptide I 1-12, two amino acids longer at the C-terminal (Leu, Gln) than peptide I 1-10 (Figure 3c). The post-translational processing of most of the peptide precursors into active neuropeptides follows a common mechanism. The processing enzymes include endopeptidases that cleave the propeptide at specific sites, usually pairs of the basic amino acids lysine and arginine, which are subsequently selectively removed from the C-terminus by the carboxypeptidase enzymes.3,4 The majority of the novel neuropeptides identified in this study are processed at the sites mentioned above. The discovery of C-terminally amidated peptides in the present report additionally supports biological activity since this type of post-translational modification predominantly occurs on bioactive neuropeptides24 (Figure 3a and b). In the hypothalamus of both rat and mouse, one of the novel peptides from the neuroendocrine protein VGF was identified with a pyroglutamic acid modification of its N-terminal Gln residue. In the rat hypothalamus, a novel peptide from the CART protein was identified carrying a phosphorylation at Ser48 that has not previously been described. Melanotropin alpha was modified both with amidation and acetylation at two sites (amidation at Val136 and acetylation at Ser124 and Ser126) as previously described in guinea pig.25 Additionally, another acetylation state of melanotropin alpha was identified in mouse carrying only one acetylation (Ser124). In future studies, we will study changes in peptide patterns (differential display peptidomics) and levels following various treatments and disorders. For these purposes, it will be 218
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important to develop approaches to quantify the levels of neuropeptides in a reproducible manner. Traditionally, peptides in nervous tissue have been studied by immunoassays. The limitations of this technique include cross-immunoreactivity with structurally similar peptides thus preventing the unequivocal identification of a specific neuropeptide, and the restriction in the number of peptides that can be analyzed simultaneously. Another common method includes measurement of mRNA expression levels. These methods have the disadvantage of not displaying changes related to posttranslational events, e.g., proteolytic cleavage and amino acid modifications,which are common features of neuropeptides. In addition, the latter method may be misleading since mRNA levels have not always been shown to be directly correlated to protein levels.26 As shown in the present study, a method requiring low amounts of brain tissue that is capable of endogenous neuropeptide detection and quantification is pertinent in neuroscience research. In conclusion, we have shown that analyzing peptides extracted from microwaved tissue using on-line nanoLC-ESIQ-TOF-MS and MS/MS is a powerful combination for simultaneous detection and identification of a large number of neuropeptides and their post-translational modifications present in the brain and thus complements standard proteomic methods.
Acknowledgment. This study was sponsored by the Swedish Research Council, Medicine, Grant No. 11565 and Natural Science, Grant No I-1179 (special program “Signals of Life”), the Swedish Foundation for International Cooperation in Research and Higher Education (STINT) Institutional Grant and the K&A Wallenberg Foundation. We are grateful to have obtained tissue from microwave sacrificed rats and mice from Dr S. Gruber and Professor A. Mathe´ at the Karolinska Institutet, Stockholm, Sweden and Professor P. Greengard at the Rockefeller University, New York. We also thank A. Kaplan, Dr. L. Bjo¨rkesten at Amersham Biosciences, Uppsala, Sweden for providing visualization software and M. Norrman at Uppsala University, Uppsala, Sweden for assistance in sequence identification of peptides. References (1) Hokfelt, T.; Millhorn, D.; Seroogy, K.; Tsuruo, Y.; Ceccatelli, S.; Lindh, B.; Meister, B.; Melander, T.; Schalling, M.; Bartfai, T. Coexistence of peptides with classical neurotransmitters. Experientia 1987, 43, 768-780. (2) Hokfelt, T.; Broberger, C.; Xu, Z.; Sergeyev, V.; Ubink, R.; Diez, M. Neuropeptides-an overview. Neuropharmacology 2000, 39, 1337-1356. (3) Benjannet, S.; Rondeau, N.; Day, R.; Chretien, M.; Seidah, N. G. PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 3564-3568. (4) Che, F. Y.; Yan, L.; Li, H.; Mzhavia, N.; Devi, L. A.; Fricker, L. D. Identification of peptides from brain and pituitary of Cpe(fat)/Cpe(fat) mice. Proc. Natl. Acad. Sci., U.S.A. 2001, 98, 9971-9976. (5) Schmidt, W. E.; Conlon, J. M.; Mutt, V.; Carlquist, M.; Gallwitz, B.; Creutzfeldt, W. Identification of the C-terminally alphaamidated amino acid in peptides by high-performance liquid chromatography. Eur. J. Biochem. 1987, 162, 467-472. (6) Tatemoto, K.; Mutt, V. Chemical determination of polypeptide hormones. Proc. Natl. Acad. Sci., U.S.A. 1978, 75, 4115-4119. (7) Tatemoto, K.; Mutt, V. Isolation of two novel candidate hormones using a chemical method for finding naturally occurring polypeptides. Nature 1980, 285, 417-418. (8) Mutt, V. Recent developments in the chemistry of gastrointestinal peptides. Eur. J. Clin. Invest. 1990, 20, 2-9.
research articles
Novel Hypothalamic Neuropeptides (9) Sko¨ld, K.; Svensson, M.; Kaplan, A.; Bjo¨rkesten, L.; Åstro¨m, J.; Andre´n, P. E. A neuroproteomic approach to targeting neuropeptides in the brain. Proteomics 2002, 2, 447-454. (10) Slemmon, J. R.; Flood, D. G. Profiling of endogenous brain peptides and small proteins: methodology, computer-assisted analysis, and application to aging and lesion models. Neurobiol. Aging. 1992, 13, 649-660. (11) Karelin, A. A.; Philippova, M. M.; Yatskin, O. N.; Kalinina, O. A.; Nazimov, I. V.; Blishchenko, E. Y.; Ivanov, V. T. Peptides comprising the bulk of rat brain extracts: isolation, amino acid sequences and biological activity. J. Pept. Sci. 2000, 6, 345-354. (12) Theodorsson, E.; Stenfors, C.; Mathe, A. A. Microwave irradiation increases recovery of neuropeptides from brain tissues. Peptides 1990, 11, 1191-1197. (13) Mathe, A. A.; Stenfors, C.; Brodin, E.; Theodorsson, E. Neuropeptides in brain: effects of microwave irradiation and decapitation. Life Sci. 1990, 46, 287-293. (14) Nylander, I.; Stenfors, C.; Tan_No, K.; Mathe, A. A.; Terenius, L. A comparison between microwave irradiation and decapitation: basal levels of dynorphin and enkephalin and the effect of chronic morphine treatment on dynorphin peptides. Neuropeptides 1997, 31, 357-365. (15) Zhu, X.; Desiderio, D. M. Methionine enkephalin-like immunoreactivity, substance P-like immunoreactivity and beta-endorphinlike immunoreactivity post-mortem stability in rat pituitary. J. Chromatogr. B Biomed. Appl. 1993, 616, 175-187. (16) Hossain, M. Z.; Murphy, L. J.; Hertzberg, E. L.; Nagy, J. I. Phosphorylated forms of connexin43 predominate in rat brain: demonstration by rapid inactivation of brain metabolism. J. Neurochem. 1994, 62, 2394-2403. (17) Lewis, R. M.; Levari, I.; Ihrig, B.; Zigmond, M. J. In vivo stimulation of D1 receptors increases the phosphorylation of proteins in the striatum. J. Neurochem. 1990, 55, 1071-1074. (18) Karelin, A. A.; Philippova, M. M.; Ivanov, V. T. Proteolytic degradation of hemoglobin in erythrocytes leads to biologically active peptides. Peptides 1995, 16, 693-697.
(19) Karelin, A. A.; Filippova, M. M.; Iatskin, O. N.; Blishchenko, E. I.; Nazimov, I. V.; Ivanov, V. T. Proteolytic degradation of hemoglobin in erythrocytes results in formation of biologically active peptides. Bioorg. Khim. 1998, 24, 271-281. (20) Ivanov, V. T.; Karelin, A. A.; Philippova, M. M.; Nazimov, I. V.; Pletnev, V. Z. Hemoglobin as a source of endogenous bioactive peptides: the concept of tissue-specific peptide pool. Biopolymers 1997, 43, 171-188. (21) Bergeron, F.; Leduc, R.; Day, R. Subtilase-like pro-protein convertases: from molecular specificity to therapeutic applications. J. Mol. Endocrinol. 2000, 24, 1-22. (22) Haskins, W. E.; Wang, Z.; Watson, C. J.; Rostand, R. R.; Witowski, S. R.; Powell, D. H.; Kennedy, R. T. Capillary LC-MS2 at the attomole level for monitoring and discovering endogenous peptides in microdialysis samples collected in vivo. Anal. Chem. 2001, 73, 5005-5014. (23) Stern, A. S.; Jones, B. N.; Shively, J. E.; Stein, S.; Undenfriend, S. Two adrenal opioid polypeptides: proposed intermediates in the processing of proenkephalin. Proc. Natl. Acad. Sci., U.S.A. 1981, 78, 1962-1966. (24) Cuttitta, F. Peptide amidation: signature of bioactivity. Anat. Rec. 1993, 236, 87-93, 172-173, 193-195. (25) Robinson, P.; Toney, K.; James, S.; Bennett, H. P. Mass spectrometric and biological characterization of guinea-pig corticotrophin. Regul. Pept. 1995, 56, 89-97. (26) Gygi, S. P.; Rochon, Y.; Franza, B. R.; Aebersold, R. Correlation between protein and mRNA abundance in yeast. Mol. Cell Biol. 1999, 19, 1720-1730. (27) Roepstorff, P.; Fohlman, J. Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed. Mass Spectrom. 1984, 11, 601.
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