Endogenous Peptide Discovery of the Rat Circadian Clock

Understanding how a small brain region, the suprachiasmatic nucleus (SCN), can synchronize the body's circadian rhythms is an ongoing research area. This important time-keeping system requires a complex suite of peptide hormones and transmitters that remain incompletely characterized. Here, capillary liquid chromatography and FTMS have been coupled with tailored software for the analysis of endogenous peptides present in the SCN of the rat brain. After ex vivo processing of brain slices, peptide extraction, identification, and characterization from tandem FTMS data with <5-ppm mass accuracy produced a hyperconfident list of 102 endogenous peptides, including 33 previously unidentified peptides, and 12 peptides that were post-translationally modified with amidation, phosphorylation, pyroglutamylation, or acetylation. This characterization of endogenous peptides from the SCN will aid in understanding the molecular mechanisms that mediate rhythmic behaviors in mammals.

pyroglutamylation, or acetylation. These aspects of peptide synthesis impact the properties of neuropeptides, further expanding their diverse physiological implications. Therefore, unveiling new peptides and unreported peptide properties is critical to advancing our understanding of nervous system function.
Historically, the analysis of neuropeptides was performed by Edman degradation in which the N-terminal amino acid is sequentially removed. However, analysis by this method is slow and does not allow for sequencing of the peptides containing N-terminal PTMs (5). Immunological techniques, such as radioimmunoassay and immunohistochemistry, are used for measuring relative peptide levels and spatial localization, but these methods only detect peptide sequences with known structure (6). More direct, high throughput methods of analyzing brain regions can be used.
Mass spectrometry, a rapid and sensitive method that has been used for the analysis of complex biological samples, can detect and identify the precise forms of neuropeptides without prior knowledge of peptide identity, with these approaches making up the field of peptidomics (7)(8)(9)(10)(11)(12). The direct tissue and single neuron analysis by MALDI MS has enabled the discovery of hundreds of neuropeptides in the last decade, and the neuronal homogenate analysis by fractionation and subsequent ESI or MALDI MS has yielded an equivalent number of new brain peptides (5). Several recent peptidome studies, including the work by Dowell et al. (10), have used the specificity of FTMS for peptide discovery (10,(13)(14)(15). Here, we combine the ability to fragment ions at ultrahigh mass accuracy (16) with a software pipeline designed for neuropeptide discovery. We use nanocapillary reversedphase LC coupled to 12 Tesla FTMS for the analysis of peptides present in the suprachiasmatic nucleus (SCN) of rat brain.
A relatively small, paired brain nucleus located at the base of the hypothalamus directly above the optic chiasm, the SCN contains a biological clock that generates circadian rhythms in behaviors and homeostatic functions (17,18). The SCN comprises ϳ10,000 cellular clocks that are integrated as a tissue level clock which, in turn, orchestrates circadian rhythms throughout the brain and body. It is sensitive to incoming signals from the light-sensing retina and other brain regions, which cause temporal adjustments that align the SCN appropriately with changes in environmental or behavioral state. Previous physiological studies have implicated peptides as critical synchronizers of normal SCN function as well as mediators of SCN inputs, internal signal processing, and outputs; however, only a small number of peptides have been identified and explored in the SCN, leaving unresolved many circadian mechanisms that may involve peptide function.
Most peptide expression in the SCN has only been studied through indirect antibody-based techniques (19 -29), although we recently used MS approaches to characterize several peptides detected in SCN releasates (30). Previous studies indicate that the SCN expresses a rich diversity of peptides relative to other brain regions studied with the same techniques. Previously used immunohistochemical approaches are not only inadequate for comprehensively evaluating PTMs and alternate isoforms of known peptides but are also incapable of exhaustively examining the full peptide complement of this complex biological network of peptidergic inputs and intrinsic components. A comprehensive study of SCN peptidomics is required that utilizes high resolution strategies for directly analyzing the peptide content of the neuronal networks comprising the SCN.
In our study, the SCN was obtained from ex vivo coronal brain slices via tissue punch and subjected to multistage peptide extraction. The SCN tissue extract was analyzed by FTMS/MS, and the high resolution MS and MS/MS data were processed using ProSightPC 2.0 (16), which allows the identification and characterization of peptides or proteins from high mass accuracy MS/MS data. In addition, the Sequence Gazer included in ProSightPC was used for manually determining PTMs (31,32). As a result, a total of 102 endogenous peptides were identified, including 33 that were previously unidentified, and 12 PTMs (including amidation, phosphorylation, pyroglutamylation, and acetylation) were found. The present study is the first comprehensive peptidomics study for identifying peptides present within the mammalian SCN. In fact, this is one of the first peptidome studies to work with discrete brain nuclei as opposed to larger brain structures and follows up on our recent report using LC-ion trap for analysis of the peptides in the supraoptic nucleus (33); here, the use of FTMS allows a greater range of PTMs to be confirmed and allows higher confidence in the peptide assignments. This information on the peptides in the SCN will serve as a basis to more exhaustively explore the extent that previously unreported SCN neuropeptides may function in SCN regulation of mammalian circadian physiology.

EXPERIMENTAL PROCEDURES
Materials-All reagents were obtained from Sigma-Aldrich unless otherwise noted. Siliconized microcentrifuge tubes (1.5 ml) were purchased from Thermo Fisher Scientific (San Jose, CA). Microcon YM-10 centrifugal filter devices were purchased from Millipore (Billerica, MA).
Animals and Circadian Time-An inbred strain of 8 -10-week-old female Long-Evans rats, LE-BluGill, demonstrated to be genetically homogeneous by high density genome scan (34) was used for these studies. Animals were fed ad libitum and were housed under constant temperature and humidity conditions in a 12:12 h light/dark cycle environment. Animals were entrained to this lighting schedule for at least 10 days prior to tissue collection. All collections of ex vivo SCN tissue samples were conducted during mid-subjective daytime ϳ6 -7 h following onset of normal lights-on conditions, referred to as Zeitgeber time (ZT) 6 -7. All vertebrate animal procedures were carried out with protocols approved by the University of Illinois at Urbana-Champaign Institutional Animal Care and Use Committee and in full compliance with National Institutes of Health guidelines for humane animal care.
Preparation of SCN Brain Punch Samples-Animal subjects were decapitated, and the brain was immediately removed from the skull. The hypothalamus was blocked, and using a mechanical tissue chopper, coronal brain sections (500-m thickness) were prepared. A brain section containing the mid-SCN was retained. A 2-mm-diameter sample corer was used to excise the paired SCN from the surrounding hypothalamus, aligning the top edge of the corer with the dorsal SCN border (see supplemental Fig. S1). This punch technique results in minimal harvest of extra-SCN hypothalamic tissue. Optic nerve tissue at the level of the optic chiasm is contained within the SCNcontaining punch. Peptidome analysis of rat optic nerve tissue produces a peptidomic profile distinct from our SCN peptidomics data (unpublished data). The SCN punch preparation was performed in glucose-/bicarbonate-/gentamicin-supplemented Earle's balanced salt solution (Invitrogen) perfused with 95% O 2 , 5% CO 2 . SCN-containing punches were immediately transferred to a siliconized microcentrifuge tube that remained submerged in powered dry ice until the time of peptide extraction.
Peptide Extraction from SCN Punches-Either 12 or 24 SCN punches were pooled and subjected to multistage peptide extraction as described in the recent work by Bora et al. (33). First, 150 or 300 l of deionized water, preheated to 90°C, was added to the SCN 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 second stage of extraction, whereas the supernatant was retained in a new microcentrifuge tube. After addition of 150 or 300 l of ice-cold acidified acetone (40:6:1 acetone/water/HCl), the sample was homogenized with ultrasonic cleaner FS30 (Thermo Fisher Scientific) for 30 s, vortexed for 1 min, and kept on ice for 1 h. The sample was vortexed again for 1 min and centrifuged at 14,000 ϫ g for 20 min at 4°C, and the supernatant was saved. Then, a third extraction was performed by adding 150 or 300 l of ice-cold 0.25% acetic acid to the tissue pellet and incubating on ice for 1 h. The acidified acetone extract was neutralized by 1 M NaOH and dried to 10 -20 l to remove the acetone. All of the extracts were combined and filtered through a Microcon centrifugal filter device (10-kDa-molecular mass cutoff). Finally, the filtered extract was concentrated using a SpeedVac and used for nanocapillary FTMS/MS injection.
Data Analysis-Resulting LC-FTMS/MS files (*.raw) were analyzed using ProSightPC 2.0 (Thermo Fisher Scientific) (16), which has several software component algorithms including cRAWler 2.0, which interprets resolved isotopic distributions based on the Xtract or thorough high resolution analysis of spectra by Horn (THRASH) algorithms. The cRAWler program first determines all precursor mass values according to user-specified tolerances such as ranges of m/z and retention time or signal-to-noise ratio and fitting parameters. The precursor and fragmentation scans corresponding to these precursors are then separately averaged and interpreted to provide a list of monoisotopic masses. This information is compiled into a ProSight upload file (.puf). In multiplexing mode, cRAWler can capture multiple precursor masses within the isolation range as multiple precursors based on an intensity cutoff (set at 10% here) relative to the base peak of the analysis window. This allows for cases where multiple precursors are fragmented together (see below).
Database Searching-Each .puf file, which typically contained hundreds of experiments from a single nano-LC-MS/MS run, was first searched in absolute mass mode (MS1 and MS2 tolerances of Ϯ10 ppm) against a database of predicted rat neuropeptides (with and without predicted modifications) generated by taking the set of known rat prohormones processed in silico via the NeuroPred algorithm (35)(36)(37). For the searches that did not identify a peptide below an E-value cutoff of 10 Ϫ4 , a search in "neuropeptide" mode was initiated against an intact rat database (UniProt 15.0, 4,318,021 protein forms) with Ϯ100-Da intact mass and Ϯ10-ppm fragment tolerance. Neuropeptide mode scans across sequences to find candidate subsequences whose masses are within tolerance of a precursor mass (no protease specificity); experimental fragment masses are then matched with theoretical fragment masses from these candidate subsequences. Neuropeptide searches along with the other mode described in this work are available through neuroProSight over the internet. A Sequence Gazer tool in neuroProSight software was used for manually determining PTMs on the peptides. The peptides identified from multiplexing mode were manually validated.

RESULTS
Two-millimeter-diameter punches of ventral hypothalamic tissue (500-m thickness) containing the bilaterally paired SCN were excised from rat coronal brain slices. At least six SCN punches, which contained ϳ360 g of total protein amount based on BCA assay, were needed for a high content nanocapillary FTMS/MS run. From a total of 10 LC-MS/MS runs for the SCN peptidome analysis, 102 endogenous peptides derived from 27 precursor proteins were identified along with 12 PTMs (amidation, phosphorylation, pyroglutamylation, and acetylation) (see Table 1). The average E-value for identification was 4 ϫ 10 Ϫ21 , 17 orders of magnitude below the conservative threshold of 10 Ϫ4 used here. This remarkable certainty of identification arises from the use of fragmentation scans with high mass accuracy and a scoring/software system that converts these data into peptide identifications with high fidelity. Thirty-three peptides (Table I, denoted with Footnote c) were not previously identified in either mouse or rat brain studies. The references for the identified peptides found in the prior studies of brain as well as SCN are included in a column of Table I. For example, the peptides derived from the prohormones gastrin-releasing peptide (GRP) and vasoactive intestinal peptide (VIP) are intrinsic SCN peptides that have received considerable attention (17, 22, 23, 25, 28, 38 -55). Surprisingly, peptides from 12 precursor proteins found in our SCN peptidome study, including cocaine-and amphetamineregulated transcript protein (CART), cerebellin-1, and proenkephalin B, were not reported in prior SCN studies. Finally, information from mRNA expression data from the mouse SCN reported in the Allen Brain Atlas (56) is included in Table I and highlights localization of prohormone synthesis for the prohormones identified from our present study. In addition to the endogenous peptides derived from prohormones, 66 peptide fragments from proteins like hemoglobin subunit ␤-1 and myelin basic protein S were also identified (supplemental Table 1). Although peptides that are protein fragments could result from post-mortem degradation during sample preparation, they may be the products of prohormone processing that are physiologically relevant. For example, small peptides formed from hemoglobin, the hemopressins, have known bioactivity and are likely enzymatically produced and are not formed during post-mortem degradation (57)(58)(59). Fig. 1 depicts the examples of FTMS and MS/MS spectra for prohormone-derived peptide forms of VIP and pituitary adenylate cyclase-activating polypeptide (PACAP) identified with E-values of 9 ϫ 10 Ϫ16 and 2 ϫ 10 Ϫ27 , respectively. Although the sequence of VIP is HSDAVFTDNYTRLRKQMA-VKKYLNSI (AA 125-152), another peptide from the VIP prohormone was identified in this study: HSDAVFTDNYTRL (AA [125][126][127][128][129][130][131][132][133][134][135][136][137]. Because the observed peptide sequence results from cleavage of the prohormone at a dibasic cleavage site (RK), it appears to be a bona fide intracellular processing product from the VIP prohormone and is not expected to arise from extracellular degradation/processing. This shortened peptide has been reported in SwePep. The observed peptide derived from the PACAP prohormone was GMGENLAAAAVD-DRAPLT (AA 111-128), whereas the previously confirmed bioactive PACAP-derived peptides are PACAP-27 (AA 131-157) and PACAP-38 (AA 131-168). Because there are dibasic residues (KR) between the observed peptide and the PACAP-27 and -38, again we assume that the observed peptide was produced from the intracellular processing of the PACAP prohormone. Fig. 2 represents the FTMS and MS/MS spectra for cerebellin (AA 57-72) and a one-amino acid-truncated form (AA 57-71), which are derived from the cerebellin-1 precursor. The two peptides co-eluted, as seen in Fig. 2A, and were identified by the data-dependent top five MS/MS acquisition strategy as seen in Fig. 2B. These two cerebellin forms were previously identified from mouse hypothalamus studies (8); however,      References found in SCN studies were for the prohormones, which were previously reported in the studies. there was no report localizing these peptides to the SCN. Interestingly, our previous work on peptide release from the rat SCN demonstrated that an unknown peak at m/z 1495.75 (MH ϩ ) changed in abundance with circadian rhythmicity over a 24-h period (30). Here, we confirm that this released peptide corresponds to a shortened form of cerebellin identified here with a 1 ϫ 10 Ϫ10 E-value. Of the 102 SCN peptides identified, 12 harbored PTMs. One example is depicted in Fig. 3, showing FTMS and MS/MS spectra for two forms of manserin, which is derived from secretogranin 2 precursor. Manserin and phosphorylated manserin were identified with E-values of 6 ϫ 10 Ϫ50 and 4 ϫ 10 Ϫ23 , respectively, and the integrated intensity values of the peptides were similar at ϳ5 ϫ 10 6 and 1 ϫ 10 6 , respectively. Phosphorylated manserin exhibited the fragment ion generated by neutral loss of H 3 PO 4 as the most prominent signal along with a few fragment ions of low abundance generated by fragmentation of the peptide backbone, which is a typical fragmentation pattern of Ser(P)/Thr(P) phosphopeptides.

Peptidomics of the Rat Suprachiasmatic Nucleus by FTMS
Finally, Fig. 4 represents a search result using multiplexed MS/MS, which resulted from use of high resolution MS/MS data and our tailored software. In Fig. 4A, the isolation of a 5 m/z region for m/z 875.79 in the FTMS scan generates two isotopic distributions, which are 1744.964 and 2623.345 Da. In the data processing of ProSightPC, the two masses were searched independently using the entire fragment ion list derived from the Fig. 4B MS/MS scan and produced the identifications of two peptides that were derived from Rhombex-40 and pro-SAAS precursors, respectively, as seen in Fig. 4C. Rhombex-40 is known as a surface adhesion  protein located at the ventral medullary surface (60); there is as yet no report of its expression in hypothalamus. The peptide big LEN, which is derived from pro-SAAS, was identified in our previous SCN studies (30). DISCUSSION Given that the SCN contains endogenous cellular oscillators that control the circadian rhythms of mammals, studying the peptides contained within the SCN is expected to increase our understanding of the circadian mechanisms. With solid-phase extraction collection strategies, we have recently analyzed the secreted peptides from the site of the SCN over a 24-h period and the released peptides from the SCN stimulated via the optic tract (30). We were able to identify several peptides previously reported by indirect studies to be present in the SCN. Furthermore, we discovered four new peptides, three of which are derived from pro-SAAS. One of the pro-SAAS-derived peptides, known as little SAAS, caused phase delays of SCN circadian rhythms in vitro.
However, there have not yet been any comprehensive peptidome studies of the SCN region using MS. Here, we performed the peptidome analysis of the rat hypothalamic SCN, which was prepared during daytime (at ZT 6), and identified 102 endogenous peptides by FTMS/MS, including 33 novel peptides. Although most of the peptides, including the novel peptides, are produced from the cleavage of classical dibasic or monobasic neuropeptide processing sites, a number of peptides have cleavage sites at Leu-Ala or Leu-Leu, which could be products of Leu-X-specific enzyme (61). There were also several peptides with unconventional cleavage sites among the newly identified peptides, for example N-terminal or C-terminal side cleavage of aspartic acid (10, 61) of the peptides from pro-SAAS and C-terminal side cleavage of tryptophan of the peptide derived from neuropeptide Y. These cleavages could occur intracellularly during prohormone processing. Alternatively, these may be occurring during extracellular processing, either endogenously or perhaps during the preparation of tissue extracts. Physiological assessments, such as we have done for little SAAS (30), are necessary to determine the functional role(s) for our novel discovery products.
Many of the identified peptides in the present study were derived from known precursors expressed in the SCN. VIP (AA 125-152), GRP (AA 24 -52), and somatostatin (AA [103][104][105][106][107][108][109][110][111][112][113][114][115][116] have been identified immunologically in neurons of the SCN core region. VIP and GRP have established roles in synchronization of the multitude of cell-based clocks in the SCN and also in relay of light information within the SCN to generate phase resetting of SCN tissue (25, 38 -41, 48 -50). In the present study, we observed shorter peptides derived from the VIP prohormone (AA 125-137) and GRP (AA 24 -41) and the other peptide fragment of somatostatin (AA 25-87). The shortened forms of VIP and GRP have been observed in mice and reported in SwePep, whereas somatostatin (AA 103-116) has not been reported. As we stated above, these shortened forms may be from processing within the vesicle or may be from extracellular peptide processing; however, the possibility of degradation during our sample processing cannot be excluded. The reasons for not detecting several expected full-length peptides may be due to short peptide lifetimes, rapid degradation, or detection limits of FTMS/MS. Of course, the prior studies involving the localization of these peptides have used immunohistochemistry and so would not distinguish the full-length and shorter peptide forms. Thus, the unusual shortened forms of these well known peptides appear to be interesting targets for follow-up functional studies. Additionally, arginine-vasopressin (AVP), well known to be released and expressed at the shell neurons of the SCN (62), was identified with the loss of its C-terminal glycine to become an amidated peptide.
In addition to the forms of known intrinsic peptides of the SCN, a PACAP-related peptide, PACAP (AA 111-128) (10,63), was identified in the present study, whereas the expected and well characterized PACAP peptides include those from AA 131-157 and 131-168. PACAP is known to be synthesized in the retina and released onto the SCN upon stimulation, transmitting photic signals via the retinohypothalamic tract (64 -76). The observation of the unique peptide in the present study could indicate that the AA 111-128 fragment has some functional role, that PACAP is synthesized in the SCN, or that there is local translation of PACAP mRNA into its prohormone.
In the present study, we also found multiple peptides that have not been previously reported in SCN circadian studies. CART, cerebellin-1, neuroendocrine protein 7B2, proenkephalin B, secretogranin 1, secretogranin 3, tachykinin 3, acyl-CoA-binding protein, brain-specific polypeptide PEP-19, PEBP-1, and dihydropyrimidinase-related proteins 2 and 3 (DRP-2 and DRP-3) precursors have not been reported in the prior SCN studies. Specifically, DRP-2 and DRP-3 are known to be expressed during neuronal ontogenesis and involve regulation of axon extension (92,93) and have not been studied in the hypothalamus. Although the peptides derived from these precursors could have functional roles, the functional studies of the peptides are beyond the scope of our study.
Our current peptidome analysis by use of high resolution data and tailored software allowed the full characterization of 12 peptides with PTMs. The phosphorylated manserin (VPSPGS*(phosphorylation)SEDDLQEEEQLEQAIKEHLGQG-SSQEMEKLAKVS) derived from secretogranin 2 precursor was identified along with unmodified manserin. Secretogranin 2 is highly expressed in the SCN of mouse (94); however, no endogenous peptides derived from secretogranin 2 have been reported in SCN studies. Recently, Beranova-Giorgianni et al. (95) performed a phosphoproteomics analysis of the human pituitary sample with trypsin digestion followed by IMAC to enrich the phosphopeptides. They observed the phosphopeptide of SPGS(*)S(*)EDDLQEEEQLEQAIK; however, they were unable to determine which Ser site was phosphorylated between the two Ser sites denoted as (*) from their study. We also detected C-terminal amidation forms of neuropeptide-glutamic acid-isoleucine, neurosecretory protein VGF precursor (LEGSFLGGSEAGERLLQQGLAQVEA-NH 2 ), melanotropin ␣, substance P, AVP, and provasopressin (FQNCPRG-NH 2 ; truncated form of AVP). Specifically, the truncated form of AVP appears not to have been reported in prior studies. An AVP fragment produced from proteolysis in the brain has been reported to be a highly potent neuropeptide (96). In addition, we identified a pyroglutamylated form of secretogranin 1 precursor (Q(pyroglutamylation)YDDGVAELDQLLHY). Al-though there is no report of this form of peptide in prior SCN studies, the homologous peptide was identified in bovine tissue adrenomedullary chromaffin vesicles (97).
In addition to peptides derived from prohormones, several peptides from non-prohormone-related proteins were detected, specifically four N-terminal acetylated forms of acyl-CoA-binding protein, brain-specific polypeptide PEP-19, thymosin ␤-4, and thymosin ␤-10. Many of these protein fragments have been reported in prior peptidome studies, and several, such as the thymosins, have been detected in SCN releasates (30), indicating that these proteins are endogenously processed into these shortened forms and may have some functional significance. Of course, others may represent sample preparation artifacts as the proteins may be degraded during tissue homogenization.

CONCLUSIONS
For identification and characterization of neuropeptides, the overall work flow described here represents a new route to discovery. Using MS/MS data with Ͻ Ͻ10-ppm mass accuracy and neuroProSight software, higher quality identification is achieved. This information allows unusual PTMs to be confirmed. The overall sensitivity of the work flow allows such assays to be made on the small nuclei in the brain. Of course, additional developments will streamline this peptide discovery process.
From a neuroscience perspective, what is particularly exciting is combining peptide discovery with approaches optimized to measure peptide release (30, 98 -101). The latter approaches provide a functional context for the peptide diversity determined here by allowing the subset of SCN peptides that are released at a particular time of day or under specific stimulation protocols to be uncovered. It is through the combination of peptide discovery and release assays that the functional implications on the complex interplay of a surprising range of peptides can be understood within the SCN.
Although we focused on analyzing the endogenous peptides present in SCN prepared at ZT 6 in the current study, the peptidome study at different ZTs can be considered as an important next step for better understanding how the SCN orchestrates circadian rhythms over a 24-h period. The SCN peptidome study at different ZTs including quantitative analysis of peptide expression is currently in progress.