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Endogenous Peptide Discovery of the Rat Circadian Clock a FOCUSED STUDY of the SUPRACHIASMATIC NUCLEUS by ULTRAHIGH PERFORMANCE TANDEM MASS □ SPECTROMETRY* S

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Endogenous Peptide Discovery of the Rat Circadian Clock a FOCUSED STUDY of the SUPRACHIASMATIC NUCLEUS by ULTRAHIGH PERFORMANCE TANDEM MASS □ SPECTROMETRY* S Research Endogenous Peptide Discovery of the Rat Circadian Clock A FOCUSED STUDY OF THE SUPRACHIASMATIC NUCLEUS BY ULTRAHIGH PERFORMANCE TANDEM MASS □ SPECTROMETRY* S Ji Eun Lee‡§, Norman Atkins, Jr.¶, Nathan G. Hatcherʈ, Leonid Zamdborg‡§, Martha U. Gillette§¶**, Jonathan V. Sweedler‡§¶ʈ, and Neil L. Kelleher‡§‡‡ Understanding how a small brain region, the suprachias- pyroglutamylation, or acetylation. These aspects of peptide matic nucleus (SCN), can synchronize the body’s circa- synthesis impact the properties of neuropeptides, further ex- dian rhythms is an ongoing research area. This important panding their diverse physiological implications. Therefore, time-keeping system requires a complex suite of peptide unveiling new peptides and unreported peptide properties is hormones and transmitters that remain incompletely critical to advancing our understanding of nervous system characterized. Here, capillary liquid chromatography and function. FTMS have been coupled with tailored software for the Historically, the analysis of neuropeptides was performed analysis of endogenous peptides present in the SCN of the rat brain. After ex vivo processing of brain slices, by Edman degradation in which the N-terminal amino acid is peptide extraction, identification, and characterization sequentially removed. However, analysis by this method is from tandem FTMS data with <5-ppm mass accuracy slow and does not allow for sequencing of the peptides con- produced a hyperconfident list of 102 endogenous pep- taining N-terminal PTMs (5). Immunological techniques, such tides, including 33 previously unidentified peptides, and as radioimmunoassay and immunohistochemistry, are used 12 peptides that were post-translationally modified with for measuring relative peptide levels and spatial localization, amidation, phosphorylation, pyroglutamylation, or acety- but these methods only detect peptide sequences with known lation. This characterization of endogenous peptides from structure (6). More direct, high throughput methods of ana- the SCN will aid in understanding the molecular mecha- lyzing brain regions can be used. nisms that mediate rhythmic behaviors in mammals. Mass spectrometry, a rapid and sensitive method that has Molecular & Cellular Proteomics 9:285–297, 2010. been used for the analysis of complex biological samples, can detect and identify the precise forms of neuropeptides with- out prior knowledge of peptide identity, with these ap- Central nervous system neuropeptides function in cell-to- proaches making up the field of peptidomics (7–12). The cell signaling and are involved in many physiological pro- direct tissue and single neuron analysis by MALDI MS has cesses such as circadian rhythms, pain, hunger, feeding, and enabled the discovery of hundreds of neuropeptides in the body weight regulation (1–4). Neuropeptides are produced from larger protein precursors by the selective action of en- last decade, and the neuronal homogenate analysis by frac- dopeptidases, which cleave at mono- or dibasic sites and tionation and subsequent ESI or MALDI MS has yielded an then remove the C-terminal basic residues (1, 2). Some neu- equivalent number of new brain peptides (5). Several recent ropeptides undergo functionally important post-translational peptidome studies, including the work by Dowell et al. (10), modifications (PTMs),1 including amidation, phosphorylation, have used the specificity of FTMS for peptide discovery (10, 13–15). Here, we combine the ability to fragment ions at ultrahigh mass accuracy (16) with a software pipeline designed From the Departments of ‡Chemistry and **Cell and Developmental Biology, §Institute for Genomic Biology, ¶Neuroscience Program, and for neuropeptide discovery. We use nanocapillary reversed- ʈBeckman Institute, University of Illinois at Urbana-Champaign, Ur- phase LC coupled to 12 Tesla FTMS for the analysis of peptides bana, Illinois 61801 present in the suprachiasmatic nucleus (SCN) of rat brain. Received, August 6, 2009, and in revised form, November 3, 2009 A relatively small, paired brain nucleus located at the base Published, MCP Papers in Press, November 10, 2009, DOI of the hypothalamus directly above the optic chiasm, the SCN 10.1074/mcp.M900362-MCP200 1 The abbreviations used are: PTM, post-translational modification; contains a biological clock that generates circadian rhythms SCN, suprachiasmatic nucleus; ZT, Zeitgeber time; .puf, ProSight in behaviors and homeostatic functions (17, 18). The SCN upload file; GRP, gastrin-releasing peptide; VIP, vasoactive intestinal comprises ϳ10,000 cellular clocks that are integrated as a peptide; PACAP, pituitary adenylate cyclase-activating polypeptide; tissue level clock which, in turn, orchestrates circadian AVP, arginine-vasopressin; GABA, ␥-aminobutyric acid; CART, co- rhythms throughout the brain and body. It is sensitive to caine- and amphetamine-regulated transcript protein; DRP, dihydro- pyrimidinase-related protein; LTQ, linear trap quadrupole; AA, amino incoming signals from the light-sensing retina and other brain acids. regions, which cause temporal adjustments that align the © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Molecular & Cellular Proteomics 9.2 285 This paper is available on line at http://www.mcponline.org Peptidomics of the Rat Suprachiasmatic Nucleus by FTMS SCN appropriately with changes in environmental or behav- homogeneous by high density genome scan (34) was used for these ioral state. Previous physiological studies have implicated pep- studies. Animals were fed ad libitum and were housed under constant tides as critical synchronizers of normal SCN function as well as temperature and humidity conditions in a 12:12 h light/dark cycle environment. Animals were entrained to this lighting schedule for at mediators of SCN inputs, internal signal processing, and out- least 10 days prior to tissue collection. All collections of ex vivo SCN puts; however, only a small number of peptides have been tissue samples were conducted during mid-subjective daytime ϳ6–7 identified and explored in the SCN, leaving unresolved many h following onset of normal lights-on conditions, referred to as Zeit- circadian mechanisms that may involve peptide function. geber time (ZT) 6–7. All vertebrate animal procedures were carried out Most peptide expression in the SCN has only been studied with protocols approved by the University of Illinois at Urbana-Cham- paign Institutional Animal Care and Use Committee and in full com- through indirect antibody-based techniques (19–29), al- pliance with National Institutes of Health guidelines for humane ani- though we recently used MS approaches to characterize sev- mal care. eral peptides detected in SCN releasates (30). Previous stud- Preparation of SCN Brain Punch Samples—Animal subjects were ies indicate that the SCN expresses a rich diversity of decapitated, and the brain was immediately removed from the skull. peptides relative to other brain regions studied with the The hypothalamus was blocked, and using a mechanical tissue chop- per, coronal brain sections (500-␮m thickness) were prepared. A brain same techniques. Previously used immunohistochemical ap- section containing the mid-SCN was retained. A 2-mm-diameter proaches are not only inadequate for comprehensively eval- sample corer was used to excise the paired SCN from the surround- uating PTMs and alternate isoforms of known peptides but ing hypothalamus, aligning the top edge of the corer with the dorsal are also incapable of exhaustively examining the full peptide SCN border (see supplemental Fig. S1). This punch technique results complement of this complex biological network of peptidergic in minimal harvest of extra-SCN hypothalamic tissue. Optic nerve inputs and intrinsic components. A comprehensive study of tissue at the level of the optic chiasm is contained within the SCN- containing punch. Peptidome analysis of rat optic nerve tissue pro- SCN peptidomics is required that utilizes high resolution strat- duces a peptidomic profile distinct from our SCN peptidomics data egies for directly analyzing the peptide content of the neuronal (unpublished data). The SCN punch preparation was performed in networks comprising the SCN. glucose-/bicarbonate-/gentamicin-supplemented Earle’s balanced In our study, the SCN was obtained from ex vivo coronal salt solution (Invitrogen) perfused with 95% O2,5%CO2. SCN-con- brain slices via tissue punch and subjected to multistage taining punches were immediately transferred to a siliconized micro- centrifuge tube that remained submerged in powered dry ice until the peptide extraction. The SCN tissue extract was analyzed by time of peptide extraction. FTMS/MS, and the high resolution MS and MS/MS data were Peptide Extraction from SCN Punches—Either 12 or 24 SCN processed using ProSightPC 2.0 (16), which allows the iden- punches were pooled and subjected to multistage peptide extraction tification and characterization of peptides or proteins from as described in the recent work by Bora et al. (33). First, 150 or 300 high mass accuracy MS/MS data. In addition, the Sequence ␮l of deionized water, preheated to 90 °C, was added to the SCN Gazer included in ProSightPC was used for manually deter- punches. The sample was boiled for 10 min and centrifuged at 14,000 ϫ g for 10 min. The resulting tissue pellet was subjected to the mining PTMs (31, 32). As a result, a total of 102 endogenous second stage of extraction, whereas the supernatant was retained in peptides were identified, including
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