Quantitative Mass Spectrometry Reveals Food Intake-Induced Neuropeptide Level Changes in Rat Brain: Functional Assessment of Selected Neuropeptides as Feeding Regulators
2017; Elsevier BV; Volume: 16; Issue: 11 Linguagem: Inglês
10.1074/mcp.ra117.000057
ISSN1535-9484
AutoresHui Ye, Jingxin Wang, Zichuan Tian, Fengfei Ma, James A. Dowell, Quentin Z. Bremer, Gaoyuan Lu, Brian A. Baldo, Lingjun Li,
Tópico(s)Receptor Mechanisms and Signaling
ResumoEndogenous neuropeptides are important signaling molecules that function as regulators of food intake and body weight. Previous work has shown that neuropeptide gene expression levels in a forebrain reward site, the nucleus accumbens (NAc), were changed by feeding. To directly monitor feeding-induced changes in neuropeptide expression levels within the NAc, we employed a combination of cryostat dissection, heat stabilization, neuropeptide extraction and label-free quantitative neuropeptidomics via a liquid chromatography-high resolution mass spectrometry platform. Using this methodology, we described the first neuropeptidome in NAc and discovered that feeding caused the expression level changes of multiple neuropeptides derived from different precursors, especially proSAAS-derived peptides such as Big LEN, PEN and little SAAS. We further investigated the regulatory functions of these neuropeptides derived from the ProSAAS family by performing an intra-NAc microinjection experiment using the identified ProSAAS neuropeptides, 'Big-LEN′ and 'PEN′. Big LEN significantly increased rats' food and water intake, whereas both big LEN and PEN affected other behaviors including locomotion, drinking and grooming. In addition, we quantified the feeding-induced changes of peptides from hippocampus, hypothalamus and striatum to reveal the neuropeptide interplay among different anatomical regions. In summary, our study demonstrated neuropeptidomic changes in response to food intake in the rat NAc and other key brain regions. Importantly, the microinfusion of ProSAAS peptides into NAc revealed that they are behaviorally active in this brain site, suggesting the potential use of these peptides as therapeutics for eating disorders. Endogenous neuropeptides are important signaling molecules that function as regulators of food intake and body weight. Previous work has shown that neuropeptide gene expression levels in a forebrain reward site, the nucleus accumbens (NAc), were changed by feeding. To directly monitor feeding-induced changes in neuropeptide expression levels within the NAc, we employed a combination of cryostat dissection, heat stabilization, neuropeptide extraction and label-free quantitative neuropeptidomics via a liquid chromatography-high resolution mass spectrometry platform. Using this methodology, we described the first neuropeptidome in NAc and discovered that feeding caused the expression level changes of multiple neuropeptides derived from different precursors, especially proSAAS-derived peptides such as Big LEN, PEN and little SAAS. We further investigated the regulatory functions of these neuropeptides derived from the ProSAAS family by performing an intra-NAc microinjection experiment using the identified ProSAAS neuropeptides, 'Big-LEN′ and 'PEN′. Big LEN significantly increased rats' food and water intake, whereas both big LEN and PEN affected other behaviors including locomotion, drinking and grooming. In addition, we quantified the feeding-induced changes of peptides from hippocampus, hypothalamus and striatum to reveal the neuropeptide interplay among different anatomical regions. In summary, our study demonstrated neuropeptidomic changes in response to food intake in the rat NAc and other key brain regions. Importantly, the microinfusion of ProSAAS peptides into NAc revealed that they are behaviorally active in this brain site, suggesting the potential use of these peptides as therapeutics for eating disorders. Neuropeptides represent the largest and most diverse class of cell-to-cell signaling molecules that modulate neurotransmission. These short amino acid chains regulate a wide range of processes such as stress, pain, addiction, memory, circadian rhythm, reproduction, and food intake (1.Schank J.R. Ryabinin A.E. Giardino W.J. Ciccocioppo R. Heilig M. Stress-related neuropeptides and addictive behaviors: beyond the usual suspects.Neuron. 2012; 76: 192-208Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 2.Podvin S. Yaksh T. Hook V. The Emerging Role of Spinal Dynorphin in Chronic Pain: A Therapeutic Perspective.Annu. Rev. Pharmacol. Toxicol. 2016; 56: 511-533Crossref PubMed Scopus (33) Google Scholar, 3.An S. Harang R. Meeker K. Granados-Fuentes D. Tsai C.A. Mazuski C. Kim J. Doyle 3rd, F.J. Petzold L.R. Herzog E.D. A neuropeptide speeds circadian entrainment by reducing intercellular synchrony.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: E4355-E4361Crossref PubMed Scopus (98) Google Scholar, 4.Navarro V.M. Kaiser U.B. Metabolic influences on neuroendocrine regulation of reproduction.Curr. Opin. Endocrinol. Diabetes Obes. 2013; 20: 335-341Crossref PubMed Scopus (58) Google Scholar). Quantitative description of the expression levels of these peptide neuromodulators can enhance understanding of the mechanisms underlying neuropeptide action, and can facilitate the discovery of novel neuropeptides that may represent effective drug-development targets in multiple neurological and psychiatric illnesses (2.Podvin S. Yaksh T. Hook V. The Emerging Role of Spinal Dynorphin in Chronic Pain: A Therapeutic Perspective.Annu. Rev. Pharmacol. Toxicol. 2016; 56: 511-533Crossref PubMed Scopus (33) Google Scholar, 5.Gautron L. Elmquist J.K. Williams K.W. Neural control of energy balance: translating circuits to therapies.Cell. 2015; 161: 133-145Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Neuropeptide actions have been studied extensively about their involvement in motivational disorders, including drug addiction, obesity, and eating disorders. Interestingly, several studies have suggested significant overlap in peptide-based mechanisms between drug addiction and the hedonistic aspects of feeding (6.Saper C.B. Chou T.C. Elmquist J.K. The need to feed: homeostatic and hedonic control of eating.Neuron. 2002; 36: 199-211Abstract Full Text Full Text PDF PubMed Scopus (889) Google Scholar, 7.Spangler R. Wittkowski K.M. Goddard N.L. Avena N.M. Hoebel B.G. Leibowitz S.F. Opiate-like effects of sugar on gene expression in reward areas of the rat brain.Brain Res. Mol. Brain Res. 2004; 124: 134-142Crossref PubMed Scopus (207) Google Scholar, 8.DiLeone R.J. Taylor J.R. Picciotto M.R. The drive to eat: comparisons and distinctions between mechanisms of food reward and drug addiction.Nat. Neurosci. 2012; 15: 1330-1335Crossref PubMed Scopus (164) Google Scholar, 9.Kelley A.E. Berridge K.C. The neuroscience of natural rewards: relevance to addictive drugs.J. Neurosci. 2002; 22: 3306-3311Crossref PubMed Google Scholar). Much of this work has focused on the actions of opioid peptides, which is understandable given the significant abuse liability of the opiate class of drugs (10.Berridge K.C. The debate over dopamine's role in reward: the case for incentive salience.Psychopharmacology. 2007; 191: 391-431Crossref PubMed Scopus (1635) Google Scholar, 11.Pecina S. Berridge K.C. Opioid site in nucleus accumbens shell mediates eating and hedonic ‘liking’ for food: map based on microinjection Fos plumes.Brain Res. 2000; 863: 71-86Crossref PubMed Scopus (289) Google Scholar, 12.MacDonald A.F. Billington C.J. Levine A.S. Alterations in food intake by opioid and dopamine signaling pathways between the ventral tegmental area and the shell of the nucleus accumbens.Brain Res. 2004; 1018: 78-85Crossref PubMed Scopus (68) Google Scholar, 13.Levine A.S. Billington C.J. Opioids as agents of reward-related feeding: a consideration of the evidence.Physiol. Behav. 2004; 82: 57-61Crossref PubMed Scopus (115) Google Scholar, 14.Zhang M. Kelley A.E. Opiate agonists microinjected into the nucleus accumbens enhance sucrose drinking in rats.Psychopharmacology. 1997; 132: 350-360Crossref PubMed Scopus (161) Google Scholar, 15.Lord J.A. Waterfield A.A. Hughes J. Kosterlitz H.W. Endogenous opioid peptides: multiple agonists and receptors.Nature. 1977; 267: 495-499Crossref PubMed Scopus (2031) Google Scholar, 16.Pert C.B. Pasternak G. Snyder S.H. Opiate agonists and antagonists discriminated by receptor binding in brain.Science. 1973; 182: 1359-1361Crossref PubMed Scopus (409) Google Scholar). A theory has emerged stating that opioids regulate motivational function by enhancing the primary rewarding properties of calorie-dense food and drugs of abuse, in part by modifying neural activity in the nucleus accumbens (NAc) 1The abbreviations used are: NAc, nucleus accumbens; HRAM, high resolution accurate mass; PTM, post-translational modification; DS, dorsal striatum; hippo, hippocampus; HT, hypothalamus; CAN, acetonitrile; MeOH, methanol; DDA, data-dependent acquisition; IT, injection times; HCD, higher-energy collisional dissociation; NCE, normalized collision energy; BCA, bicinchoninic acid; AGC, automatic gain control; UPLC, ultraperformance liquid chromatography; CCK, cholecystokinin; proTRH, prothyrotropin-releasing hormone; PACAP, pituitary adenylate cyclase-activating polypeptide; CARTPT, cocaineand amphetamine-regulated transcript protein; VGF, neurosecretory protein VGF; PENK, proenkephalin; PDYN, prodynorphin; POMC, proopiomelanocortin; PENK A, proenkephalin-A; PENK B, Proenkephalin-B; NPY, neuropeptide Y; VIP, vasoactive intestinal peptide; PC 1/3, prohormone convertase 1/3; icv, intracerebroventricular. 1The abbreviations used are: NAc, nucleus accumbens; HRAM, high resolution accurate mass; PTM, post-translational modification; DS, dorsal striatum; hippo, hippocampus; HT, hypothalamus; CAN, acetonitrile; MeOH, methanol; DDA, data-dependent acquisition; IT, injection times; HCD, higher-energy collisional dissociation; NCE, normalized collision energy; BCA, bicinchoninic acid; AGC, automatic gain control; UPLC, ultraperformance liquid chromatography; CCK, cholecystokinin; proTRH, prothyrotropin-releasing hormone; PACAP, pituitary adenylate cyclase-activating polypeptide; CARTPT, cocaineand amphetamine-regulated transcript protein; VGF, neurosecretory protein VGF; PENK, proenkephalin; PDYN, prodynorphin; POMC, proopiomelanocortin; PENK A, proenkephalin-A; PENK B, Proenkephalin-B; NPY, neuropeptide Y; VIP, vasoactive intestinal peptide; PC 1/3, prohormone convertase 1/3; icv, intracerebroventricular., a forebrain site that crucially regulates the effects of both natural and drug rewards. For example, direct intra-NAc infusion of opioid peptides (primarily those acting at the mu-opioid receptor, such as endorphins and enkephalins) markedly increases food intake. Indeed, this is among the largest drug-induced feeding effects elicited from anywhere in the brain, with rats consuming up to 400% of their normal intake of food (17.Zhang M. Gosnell B.A. Kelley A.E. Intake of high-fat food is selectively enhanced by mu opioid receptor stimulation within the nucleus accumbens.J. Pharmacol. Exp. Ther. 1998; 285: 908-914PubMed Google Scholar). Feeding-associated behavioral states also markedly influence the striatal expression of enkephalin genes. Will and coworkers found that expression of preproenkephalin mRNA in the NAc was significantly upregulated in a food-anticipation state relative to a satiety state (18.Will M.J. Vanderheyden W.M. Kelley A.E. Striatal opioid peptide gene expression differentially tracks short-term satiety but does not vary with negative energy balance in a manner opposite to hypothalamic NPY.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2007; 292: R217-R226Crossref PubMed Scopus (22) Google Scholar), suggesting that transcriptional activation of the striatal enkephalin system is linked to the motivational drive to eat. Nevertheless, mRNA expression studies can only reveal transcript-level changes that do not always reflect changes in peptide levels. Mass spectrometry (MS)-based analyses can provide this missing information, thereby validating and extending the inferences gleaned from gene expression studies. MS-based analyses can also address a common limitation of neuropharmacological and gene-expression studies of peptide neuromodulation; namely, that these approaches typically examine only one peptide or peptide family at a time (19.van den Heuvel J.K. Furman K. Gumbs M.C. Eggels L. Opland D.M. Land B.B. Kolk S.M. Narayanan N.S. Fliers E. Kalsbeek A. DiLeone R.J. la Fleur S.E. Neuropeptide Y activity in the nucleus accumbens modulates feeding behavior and neuronal activity.Biol. Psychiatry. 2015; 77: 633-641Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 20.Jureus A. Cunningham M.J. McClain M.E. Clifton D.K. Steiner R.A. Galanin-like peptide (GALP) is a target for regulation by leptin in the hypothalamus of the rat.Endocrinology. 2000; 141: 2703-2706Crossref PubMed Scopus (0) Google Scholar, 21.Griffond B. Risold P.Y. MCH and feeding behavior-interaction with peptidic network.Peptides. 2009; 30: 2045-2051Crossref PubMed Scopus (27) Google Scholar, 22.Tachibana T. Saito S. Tomonaga S. Takagi T. Saito E.S. Boswell T. Furuse M. Intracerebroventricular injection of vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibits feeding in chicks.Neurosci. Lett. 2003; 339: 203-206Crossref PubMed Scopus (70) Google Scholar). The regulation of food intake is, however, an intricate process that takes place via a complex neural network involving coregulation of multiple neuropeptides from different peptide families. Currently, relatively little information is available regarding how diverse neuropeptide families change comprehensively in response to feeding motivational states. Furthermore, cross-talk and interplay of a multitude of feeding-responsive neuropeptides, both within a specific brain region and across different brain regions, has not been examined previously. To obtain a more comprehensive analysis of feeding-associated peptide changes in the NAc and other sites, we employed a combination of cryostat dissection, neuropeptide extraction and label-free quantitative neuropeptidomics using liquid chromatography coupled with high resolution accurate mass Orbitrap mass spectrometer. This approach enables simultaneous detection of many neuropeptides from specific brain regions of interest in a high-throughput manner, and avoids several pitfalls of older methods. Historically, neuropeptides have been predominantly studied employing radioimmunoassay, immunohistochemistry, and Edman degradation (20.Jureus A. Cunningham M.J. McClain M.E. Clifton D.K. Steiner R.A. Galanin-like peptide (GALP) is a target for regulation by leptin in the hypothalamus of the rat.Endocrinology. 2000; 141: 2703-2706Crossref PubMed Scopus (0) Google Scholar). These techniques continue to be important, but recent advances in MS have provided a new and powerful analytical platform to study neuropeptides (21.Griffond B. Risold P.Y. MCH and feeding behavior-interaction with peptidic network.Peptides. 2009; 30: 2045-2051Crossref PubMed Scopus (27) Google Scholar, 22.Tachibana T. Saito S. Tomonaga S. Takagi T. Saito E.S. Boswell T. Furuse M. Intracerebroventricular injection of vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibits feeding in chicks.Neurosci. Lett. 2003; 339: 203-206Crossref PubMed Scopus (70) Google Scholar). MS-based neuropeptidomics can identify and quantifying many neuropeptides simultaneously, yielding higher throughput and specificity, whereas immuno-based approaches suffer from relatively low throughput and possible cross-reactivity among different members of the same peptide family. Furthermore, these older methods could not accurately identify and characterize the neuropeptides with additional post-translational modifications (PTMs) such as acetylation, C-terminal amidation, and N-terminal pyro-glutamic acid. MS-based neuropeptidomic analysis of mammalian tissue samples does, however, suffer from nonspecific protein degradation. This process can produce peptide contamination peaks that can mask the signals of the less abundant endogenous neuropeptides (23.Hokfelt T. Broberger C. Xu Z.Q. Sergeyev V. Ubink R. Diez M. Neuropeptides–an overview.Neuropharmacology. 2000; 39: 1337-1356Crossref PubMed Scopus (476) Google Scholar, 24.Buchberger A. Yu Q. Li L. Advances in Mass Spectrometric Tools for Probing Neuropeptides.Annu. Rev. Anal. Chem. 2015; 8: 485-509Crossref PubMed Scopus (51) Google Scholar). Previous attempts to curtail postmortem protein degradation have employed a number of methods, including the use of transgenic mice lacking carboxypeptidase E activity (25.Fricker L.D. McKinzie A.A. Sun J. Curran E. Qian Y. Yan L. Patterson S.D. Courchesne P.L. Richards B. Levin N. Mzhavia N. Devi L.A. Douglass J. Identification and characterization of proSAAS, a granin-like neuroendocrine peptide precursor that inhibits prohormone processing.J. Neurosci. 2000; 20: 639-648Crossref PubMed Google Scholar), processing samples in a boiling buffer (26.Dowell J.A. Heyden W.V. Li L. Rat neuropeptidomics by LC-MS/MS and MALDI-FTMS: Enhanced dissection and extraction techniques coupled with 2D RP-RP HPLC.J. Proteome Res. 2006; 5: 3368-3375Crossref PubMed Scopus (70) Google Scholar), focused microwave irradiation for animal sacrifice (27.Svensson M. Skold K. Svenningsson P. Andren P.E. Peptidomics-based discovery of novel neuropeptides.J. Proteome Res. 2003; 2: 213-219Crossref PubMed Scopus (206) Google Scholar), and rapid postsacrificial microwave irradiation (28.Che F.Y. Lim J. Pan H. Biswas R. Fricker L.D. Quantitative neuropeptidomics of microwave-irradiated mouse brain and pituitary.Mol. Cell. Proteomics. 2005; 4: 1391-1405Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) Microwave irradiation heat deactivates endogenous proteases and arrests postmortem protein degradation, resulting in a clean neuropeptide sample for MS analysis. Alternatively, a DenatorTM apparatus has been developed to rapidly and focally heat individual tissue samples, and has been shown to minimize nonspecific protein degradation that might convert endogenous neuropeptides to other forms, thereby greatly enhancing accurate peptide identification and quantitation (29.Petruzziello F. Falasca S. Andren P.E. Rainer G. Zhang X. Chronic nicotine treatment impacts the regulation of opioid and non-opioid peptides in the rat dorsal striatum.Mol. Cell. Proteomics. 2013; 12: 1553-1562Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 30.Sturm R.M. Greer T. Woodards N. Gemperline E. Li L. Mass spectrometric evaluation of neuropeptidomic profiles upon heat stabilization treatment of neuroendocrine tissues in crustaceans.J. Proteome Res. 2013; 12: 743-752Crossref PubMed Scopus (25) Google Scholar). Consequently, our lab has employed a neuropeptidomics workflow combining snap freezing, cryostat dissection, and snap heat stabilization using Denator. We therefore anticipate that the neuropeptides described in this study represent mostly bioactive mature neuropeptides and intermediate cleavage products with potential biological functions. The goal of the present study was to directly interrogate feeding-induced changes in endogenous peptides, using the MS-based neuropeptidomic approach described above, in four feeding-modulatory brain regions: NAc, dorsal striatum (DS), hippocampus (hippo) and hypothalamus (HT) (11.Pecina S. Berridge K.C. Opioid site in nucleus accumbens shell mediates eating and hedonic ‘liking’ for food: map based on microinjection Fos plumes.Brain Res. 2000; 863: 71-86Crossref PubMed Scopus (289) Google Scholar, 31.Kelley A.E. Baldo B.A. Pratt W.E. A proposed hypothalamic-thalamic-striatal axis for the integration of energy balance, arousal, and food reward.J. Comp. Neurol. 2005; 493: 72-85Crossref PubMed Scopus (269) Google Scholar, 32.Kanoski S.E. Grill H.J. Hippocampus Contributions to Food Intake Control: Mnemonic, Neuroanatomical, and Endocrine Mechanisms.Biol. Psychiatry. 2017; 81: 748-756Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Notably, such peptidomic changes in response to food intake have never been characterized in the NAc region previously. We analyzed tissue punches from these brain regions in two groups of rats. In the “unfed” group, food was withheld during a 2-hour period spanning the change from the light to dark cycle, a period in which rats (which are nocturnal) normally exhibit intense feeding responses and are primed to eat because of circadian factors. In the “fed” group, rats had access to food during this time, and completed a meal. This protocol mirrors the methodology used in prior studies of feeding-responsive striatal enkephalin gene expression (18.Will M.J. Vanderheyden W.M. Kelley A.E. Striatal opioid peptide gene expression differentially tracks short-term satiety but does not vary with negative energy balance in a manner opposite to hypothalamic NPY.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2007; 292: R217-R226Crossref PubMed Scopus (22) Google Scholar). Our peptidomic approach enabled the investigation of two interrelated questions: (1) Can MS-based peptide quantification methods reveal the feeding-related peptide gene-expression changes reported in previous literature, and (2) What are the coordinated changes across diverse peptide families and brain regions in fed versus unfed behavioral states? Finally, we explored the physiological relevance of novel peptides uncovered in the NAc, by synthesizing the peptides, infusing them directly into the NAc, and monitoring ethologically relevant ingestive and exploratory-like behaviors. The objective of this study is to perform comprehensive characterization of the neuropeptide changes because of food intake in four different rat brain regions from 12 fed and unfed adult male Sprague-Dawley rats by a label-free neuropeptidomics approach. 24 rats were all sacrificed 2 h after the experimental manipulation (i.e. food given or food withheld). Rat brains were immediately dissected following decapitation and the DS, hippo, HT and NAc regions were isolated as tissue punches, followed by rapid heating via DenatorTM to minimize postmortem degradation. Four punches were pooled for each aliquot of NAc and hippo (n = 3 for each group), three punches were pooled for an aliquot of HT (n = 4 for each group), and two punches were pooled for each aliquot of DS as an aliquot (n = 6 for each group) to minimize individual variation and increase the amount of neuropeptides contained in samples. Each punch was sampled from an individual animal. The sample aliquots were then processed independently, and analyzed with LC-MS/MS on an Orbitrap platform. The MS/MS data were searched against a home-built rat neuropeptide database in PEAKS Studio for peptide identification. Relative expression level changes of the identified peptides were calculated by comparing the peak areas calculated based on extracted ion chromatograms (XIC) of each peptide. Statistical significance in peptide abundance levels between the unfed and fed groups was determined by Student's t test. Following the differential analysis of neuropeptidome, we chose several ProSAAS peptides that changed significantly on feeding by performing microinjection experiments on rat NAc. Stainless-steel guide cannulae were implanted above the NAc shell for 10 Sprague-Dawley rats. After recovery from surgery, rats received intra-NAc infusions and were placed into a behavior-observation procedure to appraise the behavioral changes of rats injected with ProSAAS-derived peptides. To explore changes in behavioral patterns over time, the first hour continuous recording of bouts and duration of each behavior was divided into 12 × 5 min time-bins. These data were subjected to two-way ANOVA (treatment X time), with repeated measures for the time factor, and posthoc analyses were carried out using Tukey's test. p < 0.05 was considered as statistically significant for all experiments. Optima grade formic acid, acetonitrile (ACN), water, and methanol (MeOH) were purchased from Fisher Scientific (Pittsburgh, PA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO). Adult male Sprague-Dawley rats (total n = 24, Harlan Sprague-Dawley, Indianapolis, IN) weighing 300–400 g were maintained in a temperature- and humidity-controlled animal colony on a 12:12-h light-dark cycle (lights off at 18:00). All subjects were naive and were allowed a minimum of a week adaptation followed by 2 days of daily handling before the beginning of the experiment. Subjects had free access to normal laboratory chow (24% protein, 4% fat) and drinking water was available ad libitum. On the day of the experiment, at 17:00, food (chow) was removed from half of the subjects, while a measured amount of food was given to the other subjects (18 g/cage). The rats were then killed at 19:00, 2 h after food was given. The time point was chosen based on the previous mRNA experiment performed in the lab (18.Will M.J. Vanderheyden W.M. Kelley A.E. Striatal opioid peptide gene expression differentially tracks short-term satiety but does not vary with negative energy balance in a manner opposite to hypothalamic NPY.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2007; 292: R217-R226Crossref PubMed Scopus (22) Google Scholar). All experimental procedures were in accordance with protocols approved by the University of Wisconsin Institutional Animal Care and Use Committee. Rats were anesthetized with isoflurane and then sacrificed by decapitation. The brain was then rapidly removed (<90 s) and snap frozen in 2-methylbutane cooled by dry ice. The frozen brain was then sectioned in 300 μm thick slices on a cryostat from Leica (Wetzlar, Germany) with a compartment temperature of −15 °C. The regions corresponding to DS, hippo, HT, and NAc were removed with a 2 mm micro-punch and stored in 1.5 ml tubes at −80 °C until extraction (see Fig. 1). Tissue punches (20–30 mg per rat) were removed from −80 °C and immediately processed by Denator Stabilizor T1 tissue stabilization device (Gothenburg, Sweden) for heat stabilization. Tissue punches were placed in a Stabilizor cartridge as a pooled aliquot. The pooled aliquots were then inserted into the device, and stabilized using the fresh preserve tissue function. The stabilization process involved uniformly heating the tissue to 95 °C for 30–45 s (depending on the tissue thickness). After stabilization, tissue was removed from the cartridge, placed in the appropriate extraction solvent, and stored at −80 °C until needed. The processed tissues were then extracted into ice-cold acidified methanol (90:10:1, MeOH: water: acetic acid) as previously described (33.Ye H. Hui L. Kellersberger K. Li L. Mapping of neuropeptides in the crustacean stomatogastric nervous system by imaging mass spectrometry.J. Am. Soc. Mass Spectrom. 2013; 24: 134-147Crossref PubMed Scopus (39) Google Scholar). The samples were then homogenized manually with a glass-glass Dounce homogenizer from Wheaton (Millville, NJ). The homogenized sample was then spun at 20,000 × g for 20 min at 4 °C to remove the insoluble pellet. Protein concentration of the pellet was determined for each sample using bicinchoninic acid (BCA) assay from Pierce (Rockford, IL) and used to adjust the differences in the neuropeptide levels contained in different sample aliquots. The adjusted supernatant was decanted and then dried in a vacuum centrifuge. Extracts were resuspended in 20 μl 0.1% formic acid aqueous solution by vortexing and brief sonication. Subsequently, the reconstituted samples were purified and concentrated by C18 ZipTip (Millipore, Billerica, MA). Briefly, the C18 ZipTip was first wetted using ACN and then pre-equilibrated for sample binding with 0.1% formic acid in water. Subsequently, the tissue extract was loaded on the C18 ZipTip. After being rinsed three times with 0.1% formic acid in water, the sample was eluted with 5 μl of ACN/water/formic acid solution (50:49.9:0.1; v/v/vol). Next, the eluent was dried and resuspended in 10 μl of 0.1% formic acid in water and subjected to future LC-MS/MS analysis. Peptide extracts from each brain sample aliquot were analyzed separately. Peptide samples were dissolved in 0.1% FA before analysis. A Waters nanoAcquity ultraperformance liquid chromatography (UPLC) (Waters Corp., Milford, MA) was coupled to a Thermo Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) for LC-MS/MS analysis. Chromatographic separations were performed on a Waters BEH 130 Å C18 reversed-phase capillary column (150 mm × 75 μm, 1.7 μm). The mobile phases used were: mobile phase A consisted of water with 0.1% FA, and mobile phase B was composed of ACN with 0.1% FA. Samples were injected and loaded onto the Waters NanoACQ 2G-V/M Sym C18 (20 mm ×180 μm, 5 μm) using 100% A at a flow rate of 5 μl/min for 1 min. Then the peptides were separated using a solvent gradient of 0–10% B over 0.5 min and then 10–30% B over 70 min at a flow rate of 300 nL/min. Data-dependent acquisition (DDA) parameters recorded MS scans in profile mode from m/z 380–1500 at a resolution of 35,000. Automatic gain control (AGC) targets of 1 × 106 and maximum injection times (IT) of 100 ms were set. The 15 most intense precursor ions were selected for MS2 higher-energy collisional dissociation (HCD) fragmentation with an isolation window of 2 Da and dynamic exclusion set at 40 s. An AGC target of 1 × 105 and a maximum IT of 150 ms was selected for tandem mass acquisition. The tandem MS spectra were acquired at a resolution of 17,500 in profile mode, with normalized collision energy (NCE) set at 27, and a fixed lower mass at m/z 110. All the raw LC-MS/MS data were imported to PEAKS Studio 7.0 (BSI, Waterloo, Ontario, Canada) for peptide identification. Data processing procedures, including peak centroiding and charge deconvolution, were conducted to refine the raw data. To perform a neuropeptide search using the PEAKS DB algorithm, the enzyme was specified as none. The peptide mass tolerance was set at 10 ppm and the MS/MS mass tolerance was set at
Referência(s)