A Quantitative Peptidomic Analysis of Peptides Related to the

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A Quantitative Peptidomic Analysis of Peptides Related to the Endogenous Opioid and Tachykinin Systems in Nucleus Accumbens of Rats Following Naloxone-Precipitated Morphine Withdrawal Uwe L. Rossbach,† Anna Nilsson,‡ Maria Fa¨lth,‡ Kim Kultima,‡ Qin Zhou,† Mathias Hallberg,† Torsten Gordh,§ Per E. Andre´n,‡ and Fred Nyberg*,† Department of Pharmaceutical Biosciences, Division of Biological Research on Drug Dependence, Uppsala University, P.O. Box 591, SE-751 24, Uppsala, Sweden, Department of Pharmaceutical Biosciences, Medical Mass Spectrometry, P.O. Box 583, SE-751 23 Uppsala, Sweden, and Multidisciplinary Pain Center, Uppsala University Hospital, S-751 85 Uppsala, Sweden Received August 25, 2008

Abstract: We have applied a recently developed label-free mass spectrometry based peptidomic approach to identify and quantify a variety of endogenous peptides from rat nucleus accumbens following withdrawal in naloxoneprecipitated, morphine-dependent rats of two separate strains. We focused on maturated, partially processed and truncated peptides derived from the peptide precursors proenkephalin, prodynorphin and preprotachykinin. The expression of several identified peptides was dependent on strain and was affected during morphine withdrawal. Keywords: Peptidome • rat brain • Morphine • Opioid peptides • Tachykinins • Withdrawal

Introduction The withdrawal syndrome in human opioid addicts causes a number of well-characterized autonomic and somatic symptoms when the drug is discontinued, but it also includes emotional or affective symptoms, including dysphoria, restlessness, hyperirritability, and anxiety.1-3 These negative emotional states are considered as important factors contributing to escalation to compulsive use, maintenance of use, and relapse after periods of abstinence. Over the past decades, a number of animal models have been developed to allow studies of neuroanatomical and neurochemical substrates mediating the negative signs that emerge from withdrawal from opioids and other drugs of abuse as well. Regarding withdrawal from opiates, it is shown that both spontaneous and antagonist-precipitated opioid withdrawal result in significant signs of aversive behavior.4,5 These include allodynia to light touch, abnormal body posture, urination, diarrhea, ptosis, piloerection, rearing, headshakes, hind limb extension, writhing, grooming, chewing/teeth chattering and tremor. Each of these features is a result of adaptive changes in several neuronal networks. Among the best-characterized brain regions for the cellular adaptations that occur during prolonged activation of µ-opioid * To whom correspondence should be addressed. Fax, +46-18-501920; phone, +46-18-4714620; e-mail, [email protected]. † Uppsala University. ‡ Medical Mass Spectrometry. § Uppsala University Hospital. 10.1021/pr800669g CCC: $40.75

 2009 American Chemical Society

receptors are the locus ceruleus, amygdala, hippocampus, nucleus accumbens (NAcc), striatum, and the ventral tegmental area (VTA), where an increased neuronal activity has been observed.3 Part of this increased activity is thought to be attributable to changes in the cAMP signaling pathway and subsequent changes in protein phosphorylation but also in gene expression.6 Adaptive changes that occur in NAcc and VTA have received particular interest, as these regions constitute parts of the mesolimbic dopaminergic system, a neuronal network critical for drug reward.7,8 Here, in addition to dopaminergic circuits, the endogenous opioid peptide and the tachykinin systems have been the targets for research aiming to map the neurochemistry underlying the aversive reactions seen during opiate withdrawal. However, the access to efficient and reliable techniques has been a limiting step in this ambition. Historically, analyses of the expression of protein and peptides have often relied on a variety of immunological techniques, such as radioimmunoassay (RIA) and immunocytochemical methods. RIA procedures have been used to probe dynorphins, enkephalins as well as the tachykinin substance P (SP) and related peptides during morphine withdrawal.9-11 Alternatively, studies on protein and peptide expression have relied on inference from the characterization of mRNA changes. Molecular analysis of RNA has rapidly evolved from classical techniques such as the Northern blot to current large-scale, high-throughput microarray analyses. Some studies combine Northern blot analysis with Western blot to get a simultaneous assessment of both the protein and its gene transcript. An example of this is the study reported by Van Bockstaele and co-workers.12 However, all these techniques only detect peptide sequences or gene transcripts with known structure and would hardly detect any gene product or message not previously identified. Furthermore, even when the sequence of the precursor protein is known, it is not always evident which active sequences are released during the different physiological or pathophysiological conditions. Therefore, the access of techniques capable of resolving peptides or proteins with unknown structures is highly desirable. In recent years, the concept of peptidomics has been formulated.13-16 Peptidomics offers an alternate means of expression analysis and has evolved as direct rather than a secondary procedure for measurement of peptide expression. Journal of Proteome Research 2009, 8, 1091–1098 1091 Published on Web 01/21/2009

technical notes It combines capillary LC with modern mass spectrometry (MS). Like genomics, peptidomics has been developed to a discovery science of large scale. It emerged from being a complement to classical proteomics to a technical approach for routine measurements of peptide expression. In the present study, we have had access to a recently developed approach for investigation of neuropeptidome.15,17 Thus, a procedure to optimize the identification process for endogenous peptides analyzed by tandem mass spectrometry by improving the sequence collections used by the search engines have been applied in studies of the enkephalin and tachykinin systems. Studies were directed to search for peptide structures derived from the preproenkephalin, preprodynorphin and the preprotachykinin systems, which may be affected during opiate withdrawal in the male rat. Two different rat strains were made tolerant to morphine and withdrawal was precipitated by a naloxone challenge. Recording of withdrawal signs animals were followed by decapitation, and brain tissues were collected and processed for further analysis using a recently developed label-free mass spectrometry based peptidomic approach for the identification and quantification of endogenous peptides.

Materials and Methods Materials. Morphine hydrochloride and sodium chloride solutions (9 mg/mL) were obtained from Apoteket AB, Sweden. The opioid receptor antagonist naloxone (Naloxone hydrochloride) was purchased from Sigma-Aldrich, Sweden. Deuterated internal standards (Met-enkephalin [YGGFM F(D8)], neurotensin [pyr-(Q)LYENKPRRPYIL L(D3)] and substance P [RPKPQQFFGLM-amide F(D8)]) were synthesized by the Department of Medicinal Chemistry, Division of Organic Pharmaceutical Chemistry, Uppsala University, Sweden. Each deuterated peptide was purified using HPLC and validated by MS/ MS. All other chemicals and solvents were of analytical grade from commercial sources. Animal Experiments. Male Sprague-Dawley (SD) rats and male Wistar (W) rats weighing 230-270 g were obtained from B&K Sollentuna, Sweden. They were housed in groups of 3 to 4 rats per cage in air-ventilated rooms (humidity 50-60%, temperature 22-24 °C) under a 12 h-dark/12 h-light cycle and were given food and water ad libitum. Before the experiment, all rats were allowed to adapt to the laboratory environment for 1 week. Prior to drug or vehicle injections, each strain was randomly divided into two groups of seven rats. One group of each strain was treated with morphine and the other with saline. Rats treated with morphine received the drug by subcutaneous (sc) injections with increasing doses to avoid respiratory depression-related deaths at the beginning of the experiment. The following doses were chosen for twice daily sc injections: 2.5 mg/kg on day 1, 5 mg/kg on day 2, and from day 3 and onward injections of 10 mg/kg morphine were given to the rats until tolerance to the opiate was detected. Development of morphine tolerance was determined by assessing the tail-flick latencies using the Model 33 Tail Flick Analgesia Meter (IITC, Life Science) every second day. Rats belonging to the control group received vehicle injection (saline, sc) in an identical fashion and tail-flick latencies were also measured on every second day. On day 14, when morphine-treated rats were completely tolerant to the opiate, all animals were challenged with a single dose (2 mg/kg, sc) of naloxone as described in a preceding paper.18 Subsequent to naloxone, the animals were individually 1092

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Rossbach et al. placed into a Perspex view box with bedding and observed for a period of 30 min. During his time, behaviors associated with the expression of opioid withdrawal (wet dog shakes, teeth chattering, salivation, writhing, diarrhea, digging, face wash, grooming, rearing, escape attempts) were recorded. The experiment ended 3.5 h after the naloxone challenge by decapitation of the rats. Brains were removed and snap-frozen in -80 °C cold isopentane and placed on dry ice to freeze thoroughly. All animal procedures followed the guidelines of European Communities Council Directive (86/609/EEC) and were approved by the local ethical committee in Uppsala, Sweden. Tissue Extraction and Sample Preparation. To avoid potential postmortem protein/peptide degradation,19,20 the collected brains were dissected in frozen state at -10 °C. We have previously studied the significance of molecular sample integrity in brain peptidomics and we identified a protein fragment (stathmin(2–20)), which we now use as sample quality indicator.19 The stathmin fragment was found present in equally low levels across all samples (coefficient of variation 4.9%). The formation of stathmin(2–20) most likely occurred either after sacrificing the animal prior to freezing or/and when -80 °C cold brain was thawed to -10 °C for the purpose of slicing. Each brain was subjected to a sagittal cut to separate the left and right hemisphere. The right hemisphere was subsequently placed into a rat brain matrix (Activational System, Inc., Forterra Drive, Warren, MI) and sliced. The slices were then denaturized by heating at 95 °C for 20 s using a rapid heat transfer inactivation instrument (Stabilizor, Denator AB, Gothenburg, Sweden). This procedure has been shown to completely stop protein degradation.19 The denaturized slices were further dissected to obtain the nucleus accumbens (core and shell), while shapes of corpus callosum and the anterior commissure were utilized for orientation in the brain during dissection.21 The collected brain region was homogenized in acetic acid (0.25%) supplemented with the deuterated internal standards (Met-enkephalin [YGGFM F(D8)], neurotensin [pyr(Q)LYENKPRRPYIL L(D3)] and substance P [RPKPQQFFGLMamide F(D8)], 40 fmol/µL) by microtip sonification (Vibra cell 750, Sonics & Materials, Inc., Newtown, CT) in a fixed ratio of 7.5 µL/mg frozen tissue. The homogenate was centrifuged at 14 000g for 50 min at 4 °C and the supernatant was collected and loaded onto a centrifugal filter device (Microcon YM-10, Millipore, Bedford, MA) with a nominal molecular weight limit of 10 000 Da. After subsequent centrifugation of the filter devices at 14 000g for 45 min at 4 °C, the flow-through containing the peptides was frozen at -80 °C and kept for further analysis. Randomized Block Design of Mass Spectrometry Analysis. A reference sample of pooled material was analyzed by mass spectrometry as every fifth sample, and in-between these pools, one sample from each group of rats was analyzed in a randomized fashion. Prior to each block, a blank run (0.25% acetic acid) was carried out. Liquid Chromatography (LC). For each animal used in the experiment, an aliquot (5 µL) of the peptide mixture was analyzed in the randomized block design on a nano-LC system (Ettan MDLC, GE Healthcare, Uppsala) coupled with an electrospray Q-Tof (Waters, Manchester, U.K.) or a linear ion trap (LTQ) (Thermo Electron, San Jose´, CA) mass spectrometer for quantification and identification, respectively. The sample was injected and desalted on a precolumn (300 µm inner diameter (i.d.) × 5 mm, C18 PepMapT, 5 µm, 100 Å, LC Packings, Amsterdam, The Netherlands) at a flow rate of 10 µL/min for

Analysis of Peptides Related to Endogenous Opioid/Tachykinin 10 min. A 15-cm fused silica emitter with a 75 µm i.d. and a 375 µm outer diameter (Proxeon Biosystems; Odense, Denmark) was used as the analytical column. The analytical column was packed in-house with reverse-phased Reprosil-Pur C18AQ 3-µm resin (Dr. Maisch, GmbH; Ammerbuch-Entringen, Germany) using a pressurized packing devise (Proxeon Biosystems). As mobile phases, Buffer A (0.25% acetic acid in water) and Buffer B (84% acetonitrile and 16% of 0.25% acetic acid in water) were used. For the separation and elution of the peptides, a 40 min gradient from 3-60% Buffer B at a flow rate of approximately 180 nL/min was applied. Peptide Quantification. The quantitative analyses of the peptides samples were performed on the Q-Tof MS instrument. The mass spectrometer was calibrated according to the mass spectrometer manufacturer’s recommendations using a PEG solution (Fluka, Switzerland). MS data was collected in a continuous mode in the m/z range 300-1000 for 50 min. The raw Q-Tof MS data was converted into ASCII files using the Data Bridge module available in the MassLynx software (V3.5, Micromass) and imported into DeCyder MS2.0 (GE Healthcare). Ion peaks were automatically detected (parameters: elution time 0.3 min, typical peak resolution 10 000, accepted charge states 1-12, signal-to-noise cutoff of 4, background subtracted quantification (smooth surface)), manually verified and if necessary removed or added in the PepDetect module. Thereafter, the DeCyder MS 2.0 time alignment function was applied permitting the following: max stretch/compress, 2 min; max leader, 10%; stretch/compress penalty, 0.1. The ion peaks of the time aligned intensity maps were then matched with following tolerances: time ( 1 min and m/z ( 0.2 Da. The intensities of the matched ion peaks were exported for subsequent normalization. Prior to normalization, the data set was limited to ion peaks with retention times less than 31 min since all ion peaks of interest had eluted at this time point. Peptide Identification. Nano-LC LTQ MS/MS analyses were carried out on three samples of each group for identification of peptides. The same nano-LC system and setup as for peptide quantification was applied. Tandem MS data was acquired in a data-dependent manner and set up to continuously switch between full MS scan (m/z 300-2000), zoom scan (most intense peak in full scan) and MS/MS scan where the most intense peak can be picked twice in a time window of 40 s before placing on an exclusion list during a 150 s period. The tandem mass spectra were converted into data files using Xcalibur (Version 2.0 SR2) and further compiled into mgf files using an in-house developed script. The obtained MS/MS spectra were searched against the following sequence collections from the SwePep database;17 SwePep precursors, SwePep peptides and SwePep prediction22 using X! Tandem23,24 providing specific identification of endogenous peptides.22 The settings for the database search were the following: peptide mass tolerance of ( 2 Da; fragment mass tolerance of ( 0.7 Da; unspecific cleavage (SwePep precursor), respectively, no-cleavage (SwePep prediction); possible post-translational modifications (Nterminal acetylation, N-terminal pyro-glutamic acid of glutamine and glutamic acid, C-terminal amidation, oxidation of methionine and tryptophan, phosphorylation of serine, tyrosine and threonine). Moreover, a MASCOT25 search was performed against Swiss-Prot database with the following settings: species Rattus; unspecific cleavage; peptide mass tolerance of ( 1.5 Da; fragment mass tolerance of ( 0.7 Da; potential modifications (N-terminal acetylation, N-terminal pyro-glutamic acid of glutamine and glutamic acid, C-terminal amidation, oxida-

technical notes

tion of methionine). In addition, masses of known peptide sequences with a potential of being derived from proenkephalin, prodynorphin and protachykinin-1 in the rat nucleus accumbens were collated with the data. Positive matches were marked in lightface font in Tables 1 and 2. Normalization and Data Analysis. Normalization was conducted on the log2 transformed data as provided by DeCyderMS software. While raw intensities are often not normal-distributed, the advantage of log-transformed data is its normal distribution, typically assumed for statistics.26,27 To correct for global intensity differences between peptide runs, the data was normalized in two steps.28 In brief, a linear regression was fitted for each individual run to a median run that were constructed of all median peak values for ions that were matched in >50% of all runs. On the basis of the linear regression equation, new values were predicted for each run. A locally weighted polynomial regression (Lowess) was then fitted for each matched peptide against the run order, and the mean value across all runs were added to retain the native intensity dimension. For each matched peptide, a proportion of 0.5 neighbors (runs), weighted by their distance to the measurement, were used for controlling the smoothness of the fit. Of 28 samples, divided into four groups, the data of five individual samples were discarded after normalization. The samples were excluded if the median coefficient of variation (CV) of an individual sample deviated more than 10% from the overall median CV obtained from the same group of treatment. Further samples were excluded if only a little fraction of peak ions, due to low intensity, was successfully matched to all other samples.29 These exclusions led to the following number of samples per group: Sprague-Dawley control (n ) 6); SpragueDawley withdrawal (n ) 4); Wistar control (n ) 7) and Wistar withdrawal (n ) 6). It occurred that some peptides were not detected in each individual samples. By setting the following minimum limits regarding the number of samples in each group, similar distribution between the compared groups were assured: Sprague-Dawley control (nmin ) 5), Sprague-Dawley withdrawal (nmin ) 4), Wistar control (nmin ) 5) and Wistar withdrawal (nmin ) 5). Missing values were imputed using k-nearest neighbor method. The statistical computation of the moderated F- and t-statistics is based on linear models and empirical bayes methods used for microarray studies.30 The reproducibility of the peptide quantification was estimated using a reference sample. The reference sample consisted of pooled material from nucleus accumbens peptide extracts of the rat equally representing all four groups. It was then analyzed as every fifth sample during the MS analysis. The coefficient of variation was computed for each detected peptide in the reference samples over the MS run. Calculating the mean of all coefficient of variation resulted in 2.7 ( 0.1% (mean CV ( SEM). To ease understandability, the normalized log2 intensities were converted into linear values throughout the text and tables presented in this paper. Only alterations in peptide levels showing more than 30% up- or down-regulation were considered. Statistics of the withdrawal behavior signs were calculated by StatView 5.0.1 (PowerPC Version 1998, SAS Institute, Inc., Cary, NC) and GraphPad InStat 3.06 (GraphPad Software, Inc., San Diego CA). All behaviors are expressed as counts during the 30 min observation period except of the diarrhea, salivation and ptosis of which occurrence among the individuals within a group was monitored. For each behavior, a parametric oneway ANOVA was performed followed by Fisher’s Protected Journal of Proteome Research • Vol. 8, No. 2, 2009 1093

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Table 1. Prodynorphin-, Proenkephalin- And Protachykinin-1-Derived Neuropeptides Detected in the Nucleus Accumbens of Sprague-Dawley and Wistar Ratsa UniProt accession no.

precursor name

peptide name

P06300 P06300 P06300 P06300 P04094 P04094 P04094 P04094 P04094 P04094 P04094 P04094 P04094 P04094 P04094 P04094 P04094 P04094 P04094 P04094 P04094 P04094 P06767 P06767 P06767 P06767 P06767

Prodynorphin Prodynorphin Prodynorphin Prodynorphin Proenkephalin A Proenkephalin A Proenkephalin A Proenkephalin A Proenkephalin A Proenkephalin A Proenkephalin A Proenkephalin A Proenkephalin A Proenkephalin A Proenkephalin A Proenkephalin A Proenkephalin A Proenkephalin A Proenkephalin A Proenkephalin A Proenkephalin A Proenkephalin A Protachykinin-1 Protachykinin-1 Protachykinin-1 Protachykinin-1 Protachykinin-1

Neoendorphin alpha Dynorphin B [1-13] Dynorphin B-29 [16-29] Dynorphin A [1-17] Leu-enkephalin Met-enkephalin Met-enkephalin-Arg-Phe Met-enkephalin-Arg-Gly-Leu Met-enkephalin-Arg-Gly-Leu

Substance P 1-7 Neurokinin A Substance P C-terminal-flanking peptide

peptide sequence and localization in precursor

observed mass LTQ

theoretical mass

YGGFLRKYPK175 YGGFLRRQFKVVT233 235 SQENPNTYSEDLDV248 202 YGGFLRRIRPKLKWDNQ218 YGGFL YGGFM 263 YGGFMRF269 188 YGGFMRGL195 188 YGGFMRGL195 Oxidation (M) 201 LEDEAKEL208 220 GRPEWWM226 202 EDEAKELQ209 201 LEDEAKELQ209 198 SPQLEDEAKE207 200 QLEDEAKELQ209 198 SPQLEDEAKEL208 198 SPQLEDEAKELQ209 120 VEPEEEANGGEILA133 85 KDSSKQDESHLLA97 219 VGRPEWWMDYQ229 118 YPVEPEEEANGGEILA133 117 LYPVEPEEEANGGEILA133 58 RPKPQQF64 83 HKTDSFVGLM92 Amide (C-term) 32 SDWSDSDQIK41 58 RPKPQQFFGLM68 Amide (C-term) 111 ALNSVAYERSAMQNYE126

1609.5 573.0 876.3 915.2 945.3 960.1 960.2 1073.3 1144.3 1201.3 1257.4 1385.5 1455.3 1456.6 1465.4 1715.8 1828.7 899.3 1132.4 1179.2 1348.0 1844.9

1227.68 1569.88 1609.67 2146.19 555.27 573.23 876.40 899.43 915.43 945.47 960.43 960.44 1073.52 1144.52 1201.58 1257.61 1385.67 1455.67 1456.72 1465.64 1715.79 1828.87 899.50 1132.57 1179.50 1346.73 1844.84

166 221

a Tandem MS confirmed identities are in bold font. Masses positively matched to masses of known peptide sequences potentially deriving from proenkephalin, prodynorphin and protachykinin-1 in the rat nucleus accumbens are shown in lightface font.

Least Significant Difference (PLSD) test. The nominal occurrence data for diarrhea, salivation and ptosis was analyzed by Fisher’s exact test (two-tailed) and p-values were adjusted by the method of Bonferroni. Differences were considered significant when p < 0.05.

Results Administration of escalating doses of morphine twice daily for 10 consecutive days according to the present scheme significantly reduced body weight gain compared with vehicletreated controls. No strain-difference was observed in this regard (data not shown). Tolerance to morphine, as assessed by measuring tail flick latencies, was clearly seen in all rats treated with this drug on day 9 and was intensified during continued injection until day 14. No significant difference in the development of tolerance to the opiate between the different rat strains was observed (data not shown). Following naloxone administration, the expression of opiate withdrawal was observed in both Sprague-Dawley and Wistar rats. Behavioral studies indicated increased expression of wet dog shakes, teeth chattering, grooming, writhing, ptosis and diarrhea (Figure 1) in both strains. The expression of wet dog shakes and writhing was significantly more pronounced in Wistar rats compared to Sprague-Dawley rats. The present study focuses on peptides derived from the precursor proteins proenkephalin, prodynorphin and protachykinin-1 since these systems have been previously shown to be involved in morphine withdrawal.10,18,31,32 Quantities of the peptides in rat nucleus accumbens peptide extracts were determined using nanoLC-ESI Q-Tof-MS. Here, we present 27 1094

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peptides derived from the above named precursors. Twentytwo of these peptides were confirmed using nano-LC LTQ MS/ MS analysis followed by a search against sequence collections from the peptide database SwePep17,22 or against the SwissProt database. The masses of five out of 27 masses were matched to masses of known peptide sequences with a potential being derived from proenkephalin, prodynorphin and protachykinin-1 in the rat nucleus accumbens. Regarding peptides related to the proenkephalin system, the opioid active compounds Met-enkephalin, Met-enkephalinArg-Phe, Met-enkephalin-Arg-Gly-Leu and Leu-enkephalin were identified. In addition, the presence of several intermediate peptides residing in the proenkephalin precursor protein (e.g., SPQLEDEAKELQ, LYPVEPEEEANGGEILA, VGRPEWWMDYQ) and truncated forms thereof were confirmed (Table 1). A significant difference between levels of the Met-enkephalin, Met-enkephalin-Arg-Phe and VGRPEWWMDYQ peptides among animal groups was observed. Further, groupwise comparisons indicated that the amounts of these peptides were significantly lower in Sprague-Dawley control rats compared to Wistar control and to naloxone-treated, morphine-dependent Sprague-Dawley rats. In addition, following the naloxone challenge, the level of the identified proenkephalin sequence VGRPEWWMDYQ was significantly increased comparing morphinetreated Sprague-Dawley rats with their saline-treated controls (2.2-fold). Comparing the levels of the undecapeptide VGRPEWWMDYQ in naloxone-treated controls, a 2.8-fold difference was observed between the two strains (Table 2). Studying the peptides derived from the prodynorphin precursor, four peptides were identified (Table 1). Three of these

Analysis of Peptides Related to Endogenous Opioid/Tachykinin

technical notes

Table 2. Intensities (Linear Scale) and Statistical Analysis of Prodynorphin-, Proenkephalin- And Protachykinin-1-Derived Neuropeptides Detected in the Nucleus Accumbens of Sprague-Dawley and Wistar Ratsa

a Intensity comparisons showing 30% or more regulation are highlighted orange. Moderated F-statistics computed p-values less than 0.01 were considered as significant (underlined). Groupwise comparisons with p-values less than 0.01, calculated by moderated t-statistics are highlighted blue. (SDSN, Sprague-Dawley saline-naloxone; SDMN, Sprague-Dawley morphine-naloxone; WSN, Wistar saline-naloxone; WMN, Wistar morphine-naloxone).

peptides were mass matched (neoendorphin alpha, dynorphin B, dynorphin A), whereas one was confirmed by tandem MS (dynorphin B-29(16–29). In Sprague-Dawley rats, naloxone treatment induced significant increases in the level of alphaneoendorphin (1.5-fold) and dynorphin B (2.3-fold) in morphinedependent animals compared to those receiving saline, whereas no differences in these peptides were seen in the two groups of Wistar rats (Table 2). Comparing naloxone-challenged control rats of both strains indicated that levels of dynorphin B in the nucleus accumbens were lower in Sprague-Dawley rats (Table 2). Search for peptides related to the protachykinin precursor in the nucleus accumbens peptidome resulted in the identification of five peptides including the previously known substance P, its heptapeptide fragment substance P(1–7) and neurokinin A, all recognized for their potency in many biological systems (Table 1). Also, the C-terminal flanking peptide and the peptide SDWSDSDQIK were detected in the actual peptidome. Our study further revealed that the level of substance P was significantly increased during naloxone-precipitated withdrawal in animals of both strains, but no distinctive levels between strains (Table 2). In contrast, the substance P amino-terminal sequence, known as substance P(1–7) was decreased (0.44-fold; p ) 0.023) during naloxone-precipitated withdrawal in Sprague-Dawley rats. However, a tendency to increased levels of substance P(1–7) was seen during withdrawal in Wistar rats (1.81-fold; p ) 0.055). The protachykinin-1 derived peptide

neurokinin A and the C-terminal flanking peptide were both significantlyincreasedduringmorphinewithdrawalinSprague-Dawley, but not in Wistar rats (Table 2). In control animals, the C-terminal flanking peptide exhibited lower levels in the nucleus accumbens peptidome from Sprague-Dawley rats compared to Wistar rats. A similar observation, however not significant, was seen for neurokinin A.

Discussion In this study, we have applied mass spectrometry based peptidomics to examine levels of peptides derived from the tachykinin and opioid peptide systems in nucleus accumbens in morphine-dependent rats of two different strains during naloxone-precipitated withdrawal. We observed significant differences in several peptides both related to treatment regimen and to the difference in strain. It is evident from the present results that treatment with opioid agonists or antagonists may affect animals differently depending upon strain. The response to opioids may differ in a strain-dependent fashion.33-36 For example, the development of opiate tolerance and the expression of withdrawal to opioids may differ due to genetic aspects.33 Here, we have noted differences in the reaction to morphine withdrawal comparing Sprague-Dawley and Wistar rats. Moreover, a strain difference was confirmed in the expression of peptides in the nucleus accumbens following naloxone challenge in Sprague-Dawley rats chronically treated with morphine. The Journal of Proteome Research • Vol. 8, No. 2, 2009 1095

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Figure 1. Observation of withdrawal behaviors following naloxone administration. Values of the withdrawal behaviors wet dog shakes, writhing, teeth chattering, grooming represent mean ( SEM. One-way-ANOVA was calculated followed by Fisher’s PLSD test. The behaviors diarrhea and ptosis are expressed as occurrence among the individuals within a group. These data were statistically compared by Fisher’s exact test, adjusting the p-value according to Bonferroni. The significance levels comparing the groups are indicated: * p < 0.05; ** p < 0.01, *** p < 0.001 (nindividuals_per_group ) 7). (SD, Sprague-Dawley; W, Wistar; SN, saline-naloxone; MN, morphinenaloxone).

observed differences in peptide levels within the strain may be associated with differences in the expression of withdrawal. Among the peptide structures derived from the proenkephalin system, the well-known endogenous opioids Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg-Phe and Met-enkephalin-Arg-Gly-Leu were found to be present in the nucleus accumbens neuropeptidome. However, several sequences with unknown biopotency were also identified. Most of these were located in the C-terminal half of the entire proenkephalin sequence. The expression levels of Met-enkephalin and Metenkephalin-Arg-Phe appeared to be significantly different between control rats of both strains. While no withdrawal impact on both peptides was observed comparing Wistar rats, 1096

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enhanced levels of Met-enkephalin (p ) 0.014) and Metenkephalin-Arg-Phe (p ) 0.063) were found during withdrawal in Sprague-Dawley rats. Previous studies have reported alteration in levels of several endogenous neuropeptide in the rat brain during opioid withdrawal.37 A significant increase of Metenkephalin in rats undergoing morphine withdrawal was earlier reported by Nylander and co-workers.38 Interestingly, a similar observation to that of Met-enkephalin was made for the VGRPEWWMDYQ peptide, representing an 11 amino acid long sequence residing in the position 219-229 in the proenkephalin precursor. The identification of the VGRPEWWMDYQ sequence as an undecapeptide, strongly affected during morphine withdrawal, is of particular interest,

Analysis of Peptides Related to Endogenous Opioid/Tachykinin since this peptide represents the C-terminal part of an earlier studied extension of Met-enkephalin named BAM-18.39 BAM18, or Met-enkephalyl-RRVGRPEWWMDYQ, is distributed throughout the rat CNS and displays high affinity for both mu and kappa opioid receptors40 and is shown to produce both hyperalgesic and analgesic effects depending on the dose.41 For the N-terminal truncated Bam-18 peptide BAM-18(8-18) (i.e., VGRPEWWMDYQ) identified in our study, no biological effect has so far been described. However, its presence in the actual neuropeptidome and its altered levels during morphine withdrawal following the naloxone challenge suggest this peptide as biomarker candidate of conditions related to pain, opiate tolerance and withdrawal. Regarding the prodynorphin system, we were able to detect all the well-characterized three sequences residing in this precursor, alpha-neoendorphin, dynorphin A and dynorphin B. In addition, the C-terminal sequence (residues 16-29) of dynorphin B-29 and Leu-enkephalin was also detected in the nucleus accumbens neuropeptidome. Dynorphin B-29 was earlier found to be enzymatically released from prodynorphin42 and this peptide was also shown to induce a dose-dependent short-lasting change on the nociceptive threshold in rats.43 Both alpha-neoendorphin and dynorphin B were found to display an increased expression during withdrawal in the neuropeptidome of Sprague-Dawley rats but not in Wistar rats. Previous studies have shown that both dynorphin A and dynorphin B are increased in the nucleus accumbens during morphine withdrawal in many strains.10,38 Here we did not show any significant alteration in dynorphin A or Leu-enkephalin as a response to the withdrawal reaction. This could result from the different techniques for quantification of peptide level. Most studies reported in the literature have used radioimmunoassay for the assessment of peptide levels, and the weakness of this technique is that due to antibody cross reactivity it is difficult to discriminate peptides with similar structures. In our studies directed to the tachykinin system, in addition to the well-characterized undecapeptide substance P and its amino-terminal substance P(1–7) we identified neurokinin A, C-terminal flanking peptide and the peptide (SDWSDADQIK) residing in the N-terminal region of the precursor. Except for this latter structure, all other peptides identified in the protachykinin precursor were more or less affected in our animal studies. A significant enhancement in both neurokinin A and the C-terminal flanking peptide was observed in Sprague-Dawley rats during morphine withdrawal. The observed enhancement of substance P in both strains is in agreement with previous studies focusing on effects morphine withdrawal on the brain of guinea pigs.44 Regarding the substance P heptapeptide fragment substance P(1–7) this peptide behaved differently depending on strain. During withdrawal, substance P(1–7) showed decreased levels in Sprague-Dawley rats (p ) 0.023), whereas the opposite was seen in Wistar rats (p ) 0.055). In earlier studies, using radioimmunoassay, we did not observe any change in the nucleus accumbens level of substance P(1-7) during morphine withdrawal in Sprague-Dawley rats.11 However, as mentioned above, this might reflect the difference in the technical approach. Moreover, early studies using SpragueDawley rats to examine effects of the morphine withdrawals on protachykinin related peptides demonstrated increased substance P levels in the striatum31 and this elevation has been thought to induce enhancement of the reaction to opiate withdrawal.45 Nevertheless, the identification of these protachykinin-1 related peptides in the actual proteome and their

technical notes

alteration in the drug challenge the animals received suggest that some of these tachykinins may represent relevant markers of events related to opioid tolerance and withdrawal. It is evident from the present study that this approach for peptidomic analysis using mass spectrometric techniques is useful for identification and quantification of neuropeptides, their prestages or degradation products in brain tissues. It also allows studies of these peptides in their response to druginduced behaviors such as reaction to opiate withdrawal. Here we have focused on a specific brain area and peptide systems, both known to be associated with reward and drug dependence. As indicated above, the present results are in good agreement with earlier studies based on immunological techniques reporting alterations in opioid peptides and tachykinins during morphine withdrawal. Some of these peptides have not been considered as relevant markers for drug or behavioral responses before, for example, the truncated BAM-18 fragment or the C- terminal flanking peptide of protachykinin-1, but here, they appear as useful candidates as biomarkers. In this study, we have focused on one single brain region and a few peptide systems related to opioid reward and addiction. We are aware that these withdrawal behaviors also include several other peptide circuits and brain areas, which also deserve attention. Indeed, a recently peptidomic study carried out in order to investigate the impact of opioids on neuropeptide levels in the mice brain demonstrated an effect on peptides other than proenkephalin, protachykinin and prodynorphin.46 Generally, a final goal for attempts to identify and secure peptides as relevant markers for any behavior or disorder must be to find a metabolic stable compound, which could be detected by a simple biopsy or in an easily accessible body fluid. An important aspect, obvious from the present study and by studies from other laboratories, is that the response to drugs and effects of drugs on the neuropeptide expression is genetically dependent. We here observed that the response to a naloxone challenge in morphine-dependent rats varies dependent on the strain. Also, in control animals, some differences in the peptide level were recorded. An earlier study comparing Lewis and Fisher rats revealed lower basal levels of the proenkephalin gene transcript in the nucleus accumbens of male Lewis rats.47 Differences in the basal levels of certain neuropeptides in limbic structures between the same strains of rats have also been demonstrated.38 To our knowledge, this study is the first using a peptidomic approach for studies of strain differences in peptide expression in the rat brain. Although the effect of morphine withdrawal on the expression of the actual peptides was more pronounced in Sprague-Dawley rats compared to Wistar rats, we believe that the observed alterations are of relevance. Strain differences regarding the expression of opioid peptides during morphine withdrawal has previously been reported from studies using immunological techniques.10 However, we are aware of a need of follow-up studies to finally settle the relevance of these peptides as potential biomarkers of the studied behavior. In conclusion, we have applied a peptidomic approach based on a recently developed label-free mass spectrometry methodology to identify and quantify peptides derived from the proenkephalin, prodynorphin and the protachykinin systems. We were able to identify several known peptides, prestages and truncated forms thereof, as well as other sequences residing in these precursors in a peptidome retrieved from nucleus accumbens of two different rat strains. Quantification of these Journal of Proteome Research • Vol. 8, No. 2, 2009 1097

technical notes peptides revealed strain differences but also differences related to naloxone-precipitated withdrawal during morphine dependence. We suggest some of the identified peptides as conceivable biomarker candidates for the studied drug-induced withdrawal behavior.

Acknowledgment. The authors would like to thank Denator AB for kindly providing their Stabilizor prototype for inactivation of protein degradation. This work was supported by the Uppsala Berzelii Center for Neurodiagnostics and the Swedish Research Council (Grants 9459, 2004-3417, 521-2007-3017, 621-2007-4686) and the K&A Wallenberg Foundation. Supporting Information Available: The supplement figure displaying the MS/MS spectra used for identification of the peptides listed in Table 1 is shown. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Koob, G. F.; Wall, T. L.; Bloom, F. E. Psychopharmacology (Berlin, Ger.) 1989, 98 (4), 530–4. (2) Nestler, E. J. J. Neurosci. 1992, 12 (7), 2439–50. (3) Koob, G. F.; Le Moal, M. Neurobiology of Addiction; Academic Press Elsevier: San Diego, CA, 2006; (4) Stinus, L.; Le Moal, M.; Koob, G. F. Neuroscience 1990, 37 (3), 767– 73. (5) Ary, M.; Cox, B.; Lomax, P. J. Pharmacol. Exp. Ther. 1977, 200 (2), 271–6. (6) Maldonado, R. Neurosci. Biobehav. Rev. 1997, 21 (1), 91–104. (7) Self, D. W.; Nestler, E. J. Annu. Rev. Neurosci. 1995, 18, 463–95. (8) Bonci, A.; Williams, J. T. J. Neurosci. 1997, 17 (2), 796–803. (9) Yukhananov, R.; Zhai, Q. Z.; Persson, S.; Post, C.; Nyberg, F. Neuropharmacology 1993, 32 (7), 703–9. (10) Nylander, I.; Vlaskovska, M.; Terenius, L. Brain Res. 1995, 683 (1), 25–35. (11) Zhou, Q.; Liu, Z.; Ray, A.; Huang, W.; Karlsson, K.; Nyberg, F. Neuropharmacology 1998, 37 (12), 1545–52. (12) Van Bockstaele, E. J.; Peoples, J.; Menko, A. S.; McHugh, K.; Drolet, G. J. Neurosci. 2000, 20 (23), 8659–66. (13) Schulz-Knappe, P.; Zucht, H. D.; Heine, G.; Jurgens, M.; Hess, R.; Schrader, M. Comb. Chem. High Throughput Screening 2001, 4 (2), 207–17. (14) Clynen, E.; Baggerman, G.; Veelaert, D.; Cerstiaens, A.; Van der Horst, D.; Harthoorn, L.; Derua, R.; Waelkens, E.; De Loof, A.; Schoofs, L. Eur. J. Biochem. 2001, 268 (7), 1929–39. (15) Svensson, M.; Skold, K.; Nilsson, A.; Falth, M.; Nydahl, K.; Svenningsson, P.; Andren, P. E. Anal. Chem. 2007, 79 (1), 15-6, 1821. (16) Verhaert, P.; Uttenweiler-Joseph, S.; de Vries, M.; Loboda, A.; Ens, W.; Standing, K. G. Proteomics 2001, 1 (1), 118–31. (17) Falth, M.; Skold, K.; Norrman, M.; Svensson, M.; Fenyo, D.; Andren, P. E. Mol. Cell. Proteomics 2006, 5 (6), 998–1005.

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