neuropeptides are involved in behavioral , cognitive , and homeostatic functions throughout the brain and body . feeding behavior , circadian rhythms , sleep , learning , and memory are just a few physiological functions regulated by these biologically active peptides . neuropeptides are synthesized as larger protein precursors and undergo a series of processing steps to produce bioactive neuropeptides . processing steps include cleaving the precursor at mono- or dibasic sites , as well as numerous post - translational modifications ( ptms ) such as amidation , acetylation , pyroglutamination , and disulfide bond formation . modifications such as acetylation can be required for resistance to degradation as well as biological activity.(5 ) before the advent of modern mass spectrometry ( ms ) , neuropeptide characterization was performed by isolating and sequencing one peptide at a time using n - terminal sequencing methods such as edman degradation . after the peptide has been characterized , appropriate antibodies are created and quantitative peptide measurements performed using radioimmunoassay . although largely effective , one disadvantage of edman degradation is that peptides containing n - terminal ptms ( acetylation , cyclic glutamate or glutamine ) can not be directly sequenced . furthermore , the peptide concentration required for sequencing is typically higher than what is required for ms structural characterization . recent developments in sample separation and ms analysis have improved identification and characterization of peptides from biological samples ranging from brain structures to single cells . when analyzing complex systems such as the mammalian brain , ms has been restricted to relatively large morphological structures : as examples , an entire hypothalamus , striatum , or hippocampus . in many cases , obtaining adequate amounts of analyte requires large sample sizes or working with high concentration peptides from neuroendocrine structures such as the pituitary that have significant amounts of peptides.(18 ) postmortem degradation also becomes an issue due to proteolytic enzymes present in sample tissues . to improve sampling efficacy , enzyme deactivation has received recent attention , with studies using a variety of techniques that include using preservatives such as dihydroxybenzoic acid ( dhb),(12 ) focused microwave irradiation before decapitation or microwave irradiation after decapitation , or boiling the tissue immediately after decapitation.(9 ) the effects of protein degradation on rodent tissue have been studied using ms - based approaches . for instance , che and colleagues(7 ) found variations in the levels of some hypothalamic peptides when a 20-s delay was introduced between decapitation and microwaving , and skld and co - workers(20 ) showed that detectable neuropeptide levels greatly decreased when the time between animal sacrifice and microwaving was as brief as 1 min . although data demonstrating the effectiveness of microwave treatment in preventing peptide degradation makes inherent sense and the research is certainly of high quality , it is also surprising when considering the large body of literature on ex vivo brain slice experiments . the brain slice preparation is a well - established technique used in studies investigating mechanisms of circadian rhythmicity , learning , neuronal repair , and synaptic plasticity . furthermore , it has been shown that altered neurosecretory vesicle pools and other cell organelles recover and stay constant for hours following brain slice preparation . advantages of the method include the ability to combine functional tests of peptide release with peptidome measurements,(27 ) and to clearly delineate small , anatomically defined brain structures such as individual brain nuclei from fresh ( non heat - treated or microwaved ) samples . here , we test the ability to isolate peptides using the established physiological preparation of the fresh brain slice . we combine the aforementioned protocols for peptidomic studies of the brain with a specific focus on the supraoptic nucleus ( son ) . although the son has been intensively studied , this may be the first mammalian neuropeptidomics examination of this small , functionally and morphologically defined cluster of cells by means of mass spectrometry . in addition to the characteristic products of the son , oxytocin , and arg - vasopressin , there are many son - derived peptides colocalized within this nucleus , presumably at lower levels . in situ hybridization , and immunohistochemical and ms studies have reported several bioactive peptides in the son coming from different precursors such as secretogranin ii,(39 ) chromogranin a,(40 ) cocaine- and amphetamine - regulated transcript ( cart ) , and neuropeptide y.(43 ) however , we speculate that these peptides are a subset of the total peptides present in the son . because of its physiological importance , abundance of neuropeptides , and easy isolation , we identify and characterize the son peptidome . in analyses using liquid chromatography ( lc ) electrospray ionization ( esi ) tandem ms ( ms / ms ) as well as lc matrix - assisted laser desorption / ionization ( maldi ) ms / ms , we identify 85 peptides , a majority of which correspond to known or previously characterized peptides . moreover , 20 peptides have been unambiguously identified with ms / ms and appear not to have been reported previously . the hypothalamic - neurohypophysial system contains populations of well - defined neurosecretory cells producing cell - to - cell signaling peptides that participate in a variety of peripheral regulatory mechanisms . these cells , the magnocellular neurons ( mcns ) of the son , are part of the hypothalamic neuroendocrine system . they are relatively large for mammalian neurons ( 30 m in diameter ) and produce significant amounts of neuropeptides , which are released into the circulatory system . interestingly , mcns appear to release their peptides dendritically , making them an interesting target for cellular , and potentially even subcellular , studies on dense core vesicle targeting and release . can we adapt single cell ms to interrogate individual mcns ? because of its high sensitivity and salt tolerance , maldi ms allows analysis not only of the peptidome of a neural tissue extract , but also of a single cell . investigation of an individual neuron using single - cell maldi ms enables identification of peptides processed from the same precursor , and also determination of colocalized neuropeptides from different precursors . jimenez et al.(14 ) used 100-m diameter molluscan neurons to mass fingerprint the neuropeptides , and several have worked with smaller , 20-m insect neurons . successfully performed maldi ms on individual 810-m diameter mammalian pituitary cells and characterized a number of pro - opiomelanocortin ( pomc ) and cart - related peptides . we profile a single mcn with maldi , without introducing exogenous labels or enzymatic treatments , in order to characterize peptide precursors and their products found within this cell population . all reagents were obtained from sigma - aldrich chemical co. ( st . louis , mo ) , unless otherwise noted . water was prepared by a milli - q filtration system ( millipore , bedford , ma ) . the animals and sacrifice procedures used are delineated in greater detail elsewhere.(48 ) briefly , 612 week old long - evans / blugill male and female rats ( university of illinois at urbanachampaign ) were sacrificed by decapitation in accordance with protocols established by the university of illinois institutional animal care and use committee and in accordance with all state and federal regulations . the brain was quickly removed and the hypothalamic coronal brain slices ( 500 m ) containing the paired son were prepared as previously reported.(23 ) slices were maintained in earle s balanced salt solution , ph 7.2 , at 37 c supplemented with 24.6 mm glucose , 26.2 mm sodium bicarbonate , and 5 mg / l gentamicin and saturated with 95% o25% co2 . brain slices were transferred to physiological phosphate buffered saline ( pbs ) and the son ( 1 mm of brain tissue ) was removed from the ex vivo coronal brain slice by punching a 1-mm plug or cutting out the nucleus on the surface of a plastic petri dish placed on a bed of ice . because the son is situated directly adjacent to the optic tracts , these structures served as easily identifiable landmarks for son collection . after the son punches were collected , the pbs was removed and the tissue processed for peptide extraction . sons from both hemispheres from 1217 animals were pooled and collected in 1.5-ml plastic vials . to increase the number of peptides extracted and eventually detected with ms , tissue extraction was performed in multiple stages with each experiment repeated at least three times . during the first stage of peptide extraction , 300 l of sterile deionized ( di ) water ( milliq ) , preheated to 90 c , was added to the son tissue samples , capped , and placed into a boiling water bath for 10 min . next , the samples were centrifuged ( mini centrifuge , model c1301 , denville , woodbridge , nj ) , the supernatant was transferred to a new vial , and the resulting pellets were frozen at 80 c until the second stage of extraction . the second stage extraction was processed using 300 l of acidified acetone ( 40:6:1 acetone / water / hcl ) for 1 h at 4 c . the sample was vortexed for 1 min and centrifuged at 14  000 rpm for 5 min , and the supernatant was collected . the third extraction stage used 300 l of 0.25% acetic acid and followed the same sample processing as stage two . all extracts were dried using a speedvac concentrator ( thermo scientific , san jose , ca ) and reconstituted with 10 l of solvent a ( aqueous 5% acetonitrile ( acn ) containing 0.1% formic acid ( fa ) and 0.01% trifluoroacetic acid ( tfa ) . after reconstitution , the three samples were pooled and filtered through a 10 kda molecular weight cutoff tube ( millipore ) ( see supporting information , figure s1 ) . chromatographic separation of the extracted peptides and mass spectrometric analyses were performed using a caplc ( micromass , u.k . ) lc system coupled with an hct ultra - ptm discovery system ion - trap mass spectrometer ( bruker daltonics ) equipped with an electrospray ionization source . for lc - maldi - time - of - flight ( tof / tof)-ms analysis , the caplc was coupled with a robotic fraction spotter ( proteineerfc , bruker daltonics ) . the samples were loaded onto a trap column ( pepmap , c18 , 5 m , 100  , dionex , sunnyvale , ca ) using a manual injector ( valco instruments co. , inc . , houston , tx ) and washed for 5 min . the trapped peptides were then eluted in reverse direction onto a reverse phase capillary column ( lc packings , 300-m inner diameter  15 cm , c18 pepmap100 , 100  ) using a solvent gradient at 2 l / min flow rate . the solvent gradient was generated using solvent a and solvent b ( aqueous 95% acn containing 0.1% fa and 0.01%tfa ) . the 70-min gradient run for lc separation included three steps : 580% solvent b in 1555 min ( linear ) ; 80% solvent b for 5560 min ( isocratic ) ; 805% solvent b in 6065 min ( linear ) . the ms data acquisition and the subsequent ms / ms of selected analytes were performed in a data - dependent manner using the esquire software ( bruker daltonics ) . for each ms scan , three peptides were selected to be fragmented , for 300500 ms , based on their charge ( preferably + 2 ) and intensity . dynamic exclusion of previously fragmented precursor ions was set to 2 spectra for a period of 60 s. the ms and ms / ms scans were performed in the range of m / z 3001500 and 502000 , respectively . for lc - maldi - tof / tof - ms analysis , chromatographic separation was performed using a 45-min gradient run comprising the following parameters : 580% solvent b in 1030 min ( linear ) ; 80% solvent b for 3035 min ( isocratic ) ; 805% solvent b in 3545 min ( linear ) . the chromatographic elutant was monitored using a uv detector ( waters , milford , ma ) and subsequently spotted onto a ground steel maldi sample plate at the rate of 60 s / spot using proteineerfc . immediately after spotting , 1 l of -cyano-4-hydroxy cinnamic acid matrix was applied to each spot . for lc - fourier transform ( ft ) ms analysis , separations were accomplished using a proteopep ii c18 column from new objective ( woburn , ma ) , with an inner diameter of 75 m and length of 10 cm . the chromatographic gradient was performed using a 1d nanolc pump from eksigent technologies ( dublin , ca ) operating at 300 nl / min with the following gradient parameters : 10% solvent b for 010 min ( isocratic ) ; 1030% solvent b for 1020 min ( linear ) ; 30100% solvent b for 2080 min ( linear ) ; 805% solvent b for 80100 min ( linear ) . peptides were detected on a 7 t linear trap quadrupole ( ltq)-ft instrument from thermo scientific , and a setting of 15 v was used for mild ion activation in the esi source . the ltq - ftms method consisted of three scan events , all in profile mode with four microscans used per full scan . an initial , full scan event was performed at a high resolution ( 100  000 ) in ft mode . the second scan event was a data - dependent collision - activated dissociation ( cad ) ms / ms scan of the most abundant peak in the previous full scan , again with ft mode detection ( 50  000 resolution ) . ms / ms settings were as follows : isolation width = 4 m / z ; minimum signal threshold = 50  000 counts ; normalized collision energy ( nce ) = 40% ; duration = 200 ms , and q - factor = 0.5 . dynamic exclusion was used for a duration of 6 min per precursor to avoid redundant fragmentation data sets . the third scan was a full scan detected in the ion trap ( low resolution ) . all neuropeptide identifications for this system were accomplished using prosightpc software ( thermo scientific ) as described below . the intact and cad fragmentation data were used to search a database of predicted neuropeptides . each sample spot was analyzed by a bruker ultraflex ii tof / tof instrument with a delayed ion extraction and a frequency tripled nd : yag laser operating at 1064 nm and 50 hz ( bruker daltonics ) . mass spectra were accumulated over 2 to 3 individual acquisitions of 300 laser shots each . intense peaks from the ms scan of samples were manually analyzed and selected for further analysis using the ms / ms feature of the ultraflex ii instrument . several peaks were assigned based on accurate mass matches without ms / ms data , normally with the confidence of these assignments enhanced by the detection of other related peptides from the same prohormone . individual son neurons and small groups of cells were isolated in accordance with the detailed protocol described previously.(17 ) briefly , brain sections were immersed in solution containing 33% glycerol and 67% of modified gey s balanced salt solution ( mgbss , in mm , 1.5 cacl2 , 4.9 kcl , 0.2 kh2po4 , 11 mgcl2 , 0.3 mgso4 , 138 nacl , 27.7 nahco3 , 0.8 na2hpo4 , 25 hepes and 10 glucose , ph 7.2 ) and kept in this solution during all isolation steps . addition of glycerol stabilizes cellular structures during tissue and cell manipulation as well as prevents dehydration of the specimen . small , several - hundred micrometers in diameter , regions of the son were removed from the slices and placed on a glass slide positioned on an inverted light microscope ( see figure 3a ) . under visual control , fine - tipped glass needles were used to isolate , pick up , and transfer individual neurons and small groups of cells onto a metal maldi sample plate . pipettes pulled from borosilicate glass were used to deposit maldi matrix - saturated dhb in water onto specimens . ( a ) photomicrograph of a neuron isolated manually from a glycerol - treated brain slice . ( b ) mass spectra acquired from individual mcn neurons using saturated dhb matrix : top trace , mass spectrum from a neuron isolated from a brain slice treated with glycerol ; middle traces , individual neuron spectra ; bottom trace , a group of son cells . tandem ms data obtained from the esi - ms / ms and maldi - tof / tof systems were selected and exported to the biotools software program ( bruker daltonics ) for further analysis . the data were converted to a mascot ( matrix science , london , u.k . ) generic file format and searched against mouse and rat ipi databases using peaks software ( bioinformatics solutions , inc . , search parameters for the peaks software included unsuspected cleavage site , n - terminal pyro - glutamic acid , and c - terminal amidation . subsequently , the search results were confirmed by manual de novo analysis using the data analysis software ( bruker daltonics ) . complimentary searches were also performed with mascot using the msdb database and an in - house database of neuropeptide prohormones . none ,  and variable modifications were considered , such as acetylation of n - terminal , amidation of c - terminal , pyro - glu of q and pyro - glu of e. the peptide mass tolerance for ms / ms data was set at 0.5 da . the ms / ms data were manually verified to contain at least three consecutive amino acid fragment ions for confirming positive identification of a peptide . in addition , maldi ms data were analyzed by direct comparison of measured molecular weights of the putative neuropeptides to the neuropred prediction engine ( http://neuroproteomics.scs.uiuc.edu/neuropred.html ) using an in - house - generated database of known rat prohormones . data files ( * .raw ) were reduced to a prosightpc upload format file ( * .puf ) using an in - house program ( crawler ) , which groups ms / ms data from all precursors of the same protein into one experiment . an experiment consisted of one precursor mass and all fragment ion masses derived from it . within this program , molecular weights of precursor and product ions were also determined using a highly modified thrash algorithm.(51 ) fragment ions for all ms / ms scans were required to have an s / n ratio of 5:1 or greater to be considered for database searching . each * .puf file typically contained hundreds of experiments from a single lc - ftms run . the experiments within these files were then searched against two types of databases using prosightpc ( thermo scientific ) and/or neuroprosight ( http://neuroprosight.scs.uiuc.edu/ ) , a highly annotated mouse proteome database of intact proteins , and a database of predicted mouse neuropeptides generated by the neuropred tool described above . first , an absolute mass search was done to find any proteins / peptides that were identified as they appeared in the database . second , a biomarker search was done to retrieve any endogenously cleaved pieces of proteins / peptides in the databases . in all cases , all retrieved proteins and peptides needed to meet two requirements to be considered a positive identification : ( 1 ) an expect score of less than 10 and , ( 2 ) no unexplained mass shifts between the observed and theoretical intact masses.(54 ) identifications that could not be assigned unambiguously were validated using the sequence gazer tool to account for errors in determination of the monoisotopic mass of the protein . peptidomics studies provide detail on the peptides present in entire brains to selected brain regions . this study represents a downscaling of sample sizes for mammalian neuropeptidomics analysis to the level of an identified nucleus , here the hypothalamic son . in comparison with previous neuropeptidomics methods devised for larger brain regions , because the ability to work with select , anatomically well - defined nuclei requires precise registration of the samples , it is easier to work with intact brain slices than heat - treated or microwaved tissue , especially as the anatomical features required for identification of the region may be altered by such treatments . one question of particular interest was whether or not neuropeptides would be detected in such samples , given that prior work showed a large reduction in neuropeptide levels and an increase in peptide fragments from proteins shortly after decapitation . our results demonstrate that , even if peptides are released and degraded immediately after death , the neurosecretory vesicles and peptide levels recover . after sampling the son by tissue punch from a brain slice increasing sample temperature by heating with 90 c di water reduces protease activity and serves as an extraction solution . we see almost no difference between extracting peptides using di water extraction and an acidified acetone extraction ( supporting information , figure s1 ) ; however , fewer peaks were seen in the acetic acid extraction . although there is variation in the data between extraction solutions , each approach produced reproducible mass spectra . because some peptides are extracted more efficiently using different conditions , our final method involves treating the son tissue with a sequential series of solvent extractions . by increasing the tissue temperature in water , followed by two - stage extraction using acidified acetone and acetic acid , we detect 20 peptides characterized using ms / ms that were not reported in prior hypothalamus peptidome studies ( table 1 ) . the quality of mass spectra ( supporting information , figure s1 ) and the number of products detected ( supporting information , table s1 ) indicate that the peptides are not affected by the lack of immediate microwaving and the delay between slice preparation and sample extraction . tandem ms data was obtained in all cases except for the last column , indicating maldi tof data . , we characterized and identified 85 peptides , many of which include peptides derived from known prohormones ( see tables 1 and 2 ) . che et al.(7 ) reported 41 neuropeptides from three brain regions ( hypothalamus , hippocampus , and striatum ) using a quadrupole time - of - flight mass spectrometer equipped with a capillary / nano - lc system . dowell and co - workers(9 ) detected 56 neuropeptides from the rat hypothalamus and striatum using a different extraction protocol and 1d lc - ms / ms , 2d lc - ms / ms , and maldi - ftms . our approach uses a smaller brain region from ex vivo brain slices and multistage extraction protocols along with lc - maldi ms , lc - esi ms , and lc - esi ftms , resulting in the identification of several unreported peptides . the results are consistent with previous studies that characterized peptides in larger brain regions discussed above . period ( . ) indicates cleavage sites . tandem ms data was obtained in all cases except for the last column , indicating maldi tof data . the use of multiple ms - based platforms , lc - maldi ms , lc - esi ms , and lc - esi ftms , enhances peptide and protein coverage as each platform excels at specific types of samples and analytes . therefore , their combination facilitates detection of larger numbers of peptides from the son samples . a majority of detected peptides have been previously observed and characterized in different parts of the mammalian brain such as the hypothalamus , striatum , pituitary , and hippocampus . our data enhances prior peptidome studies on the hypothalamus and striatum.(9 ) as previously reported peptides from these regions , we also observe the provasopressin[2432 ] ( avp ) , the pyro - glu form of cart ( figure 1a),(10 ) pomc[124136 ] ( melanotropin alpha ) , somatostatin[103116 ] ( somatostatin 14 ) , somatostatin[89100 ] , proenkephalin a[100104 ] ( met - enkephalin ) , proenkephalin a[219229 ] , proenkephalin a[198209 ] , secretogranin 1[585594],(9 ) prosaas[245260 ] ( big len ) , prosaas[4259 ] ( little saas ) and promch[131143].(9 ) figure 1 shows the ms / ms fragmentation spectra of the known peptides cart[2836 ] ( figure 1a ) and provasopressin[154168 ] ( figure 1b ) identified in the son , with figure 2 showing ms / ms results from two ms platforms . in addition to products from known prohormones , several different protein fragments have been identified in this analysis , most of which correspond to pieces of myelin basic protein ( mbp ) , the second most abundant protein in the brain ( table 2).(55 ) maldi - tof / tof ( ms / ms ) data allows the identification of several peptides in the son sample : ( a ) cart[2836 ] , a.pqedaelqpr.a , m / z 1067.52 ( which was previously characterized in a mouse brain sample ) ; ( b ) a novel peptide from the vasopressin prohormone : provasopressin[154168 ] l.agtqesvdsakprvy , m / z 1606.69 . sequencing of known peptides from somatostatin ; somatostatin[89100 ] r.sansnpamapre.r , m / z 1243.56 , using two tandem ms approaches : ( a ) maldi - tof / tof and ( b ) lc - esi ion trap . twenty novel peptides ( table 1 ) are identified using ms / ms from various precursors : provasopressin , somatostatin , secretogranin 1 , secretogranin 3 , proenkephalin b , cortistatin , somatotropin , prosaas , and neurotensin . figure 1b shows an example of the tandem ms ( ms / ms ) fragmentation spectra for a novel peptide derived from provasopressin . this peptide presents an unconventional cleavage site at leuala , a potential product of leu - x specific protease.(7 ) a majority of sequenced peptides in the son are generated by enzymatic cleavage of mono- or dibasic sites of the corresponding prohormones . however , in several cases of the newly discovered peptides , the prohormone cleavage occurs at uncommon processing sites : c - terminus of provasopressin , somatostatin , n - terminus of secretogranin i , n - terminus of secretogranin iii , n - terminus signal peptide of cortistatin , n - terminus of proenkephalin b , prosaas ( table 1 ) . the possibility that these cleavages occur due to extracellular peptide degradation in situ can not be ruled out . the peptidome of the son is as complex as the previously reported peptidome of the entire hypothalamus . at what stage of sample reduction individual mcn were manually isolated  without enzymatic treatment or in vivo or in vitro labeling of the son  using coronal brain slices stabilized with glycerol ( figure 3a ) . here , maldi ms of a small group of mcns provides a cleaner spectrum as compared with the mass profile of single cells , which is not surprising considering the significant increase in the amount of peptides available for analysis . using ms measurements , avp ( m / z 1083.45 ) can be unambiguously identified in a low mass range ( figure 3 ) and neurophysin ii ( m / z 9842.29 ) was identified in the higher mass range ( supporting information , figure s2 ) , corroborating previous reports of neurophysin prohormones being present in mcn neurons . different peaks are visible in the higher range mass spectra ( supporting information ) . because the amount of material in an individual cell is too small for ms / ms approaches , we assign these peaks based on sequenced peaks from the son study and the mass match within the individual mcn . interestingly , using such mass matching , we identified a cart - related peptide , cart[2836 ] ( figure 3b ) . reduction of sample size to the single cell level reduces the depth of peptide coverage but provides a more streamlined linkage of functional , morphological and biochemical information . for the current neuropeptidome study of the son , hypothalamic coronal brain slices kept in an oxygenated media are used and a complement of peptides detected . how can this be reconciled with prior work showing that tissue denaturation immediately after animal death is required , or large - scale protein and peptide degradation is observed ? the prior peptidome comparisons have been between tissue that is heat- or microwave - treated with various time delays , and not with living brain slices . in fact , peptide - containing dense core vesicles of neurosecretory cells in brain slices lose a fraction of their content initially ; however , the contents recover and stay constant for at least 9 h , as shown by hatton et al.(25 ) furthermore , it has been well - established that living brain slices can be used to successfully study the suprachiasmatic nucleus ( scn ) , the master biological clock . in this case , the experiments demand the maintenance of active tissue for longer than 24 h. similarly , chen et al.(56 ) found that pituitary adenylate cyclase activating peptide ( pacap ) remained active in scn brain slices longer than 19 h in continuously perfused chambers . on the basis of these studies , and with the information in prior peptidome studies , we infer that the releasable pool of peptides is rapidly depleted but recovers before the son is isolated . once the son punch is made and the tissue is removed from the chamber , degradation will occur ; we stop this process by using a hot water treatment that also serves to facilitate the peptide extraction processes . comparison of the number of characterized son peptides with the number of identified peptides reported for larger brain structures supports the efficiency of our strategy . we used a combination of mass spectrometric approaches ( lc - maldi , lc - esi , and lc - esi - ftms ) to increase the coverage in this proteomics investigation and unambiguously identify the peptides . lc - ftms / ms is a great tool for unambiguously identifying peptides through high - resolution measurements of precursor and product ions . the major drawback to this technique is the lower sensitivity inherent to ft - icr mass spectrometry . thus , it is a great tool for making peptide assignments when the ions signals are sufficient , but it trails other techniques in terms of the number of peptides identified . the detected peptides are the result of processing of known prohormones , with these processing steps either being intracellular ( before peptide release ) or extracellular ( after peptide release ) , the latter possibly being the result of degradation during sample preparation . a number of detected peptides originate from unconventional cleavage sites , which may be due to extracellular enzymes . in the case of interleukin-3[5462 ] , the removal of the c - terminal asp residue may be due to the acidic extraction . peptides originating from provasopressin precursor indicate that multiple unconventional , nonspecific cleavages may occur either during the acidic extraction or through enzymatic processing . because it is difficult to directly relate peak heights of structurally distinct peptides to the amount of peptide present ( at least without using calibration standards ) , it is difficult to know whether some of these unusually processed peptides are present at trace levels compared to the known biologically active peptides . surprisingly , one peptide that we did not detect is oxytocin , even though it is in an ideal mass range ( m / z 1006.44 ) for characterization . it should be noted that , when the entire hypothalamus was extracted , oxytocin was not reported in at least one prior study.(9 ) nevertheless , there are reports of oxytocin in the hypothalamus identified by mass(6 ) as well as following n - terminal modification.(8 ) considering that 90% of the content in the son peptides is released dendritically and not somatically or axonally , oxytocin is expected in the son . two novel peptides , cortistatin [ 2033 ] , and provasopressin[154168 ] , and a previously characterized peptide , provasopressin[151168 ] , have leu - x or leuleu cleavage sites . che et al.(7 ) assumed the presence of a leu - specific enzyme in the processing of corticotropin - like intermediary peptide of pomc prohormone , and hummon et al.(57 ) reported leuleu cleavage sites in aplysia californica prohormones . it is known that in vertebrates , the enzyme renin specifically cleaves leuleu sites.(58 ) another enzyme , chymotrypsin , previously characterized in the human , rat , and pig , cleaves leuala sites.(59 ) on the basis of these studies , we assume that cortistatin[2033 ] and provasopressin[154168 ] are cleaved by either rennin - like or chymotrypsin - like enzymes . we further speculate that provasopressin[151168 ] and provasopressin[154168 ] are processed in intracellular secretory vesicles since they also were observed in prior microwaved samples ( data not shown ) . according to che et al.,(7 ) rapid microwave irradiation before decapitation prevents vesicle fusion and neuropeptide release ; hence , the peptides remain in secretory vesicles and are available for extraction . ms / ms is capable of characterizing the ptms present in neuropeptides , which is important as these ptms often directly modify the activity or lifetime of the peptide . the most common ptms in mammalian tissues are n - terminal acetylation and c - terminal amidation.(2 ) in 1987 , fischer and spiess(60 ) identified a glutaminyl cyclase converting enzyme in mammalian brain responsible for the formation of n - terminal pyro - glu . additionally , in situ and immunocytochemical studies showed that the son contains a glutaminyl cyclase - converting enzyme required to create pyro - glu forms of several peptides . here , we detect pyro - glu forms of cart[2836],(10 ) neurotensin precursor[150164 ] , and progonadoliberin i precursor[2433 ] . we also detect amidation of avp , substance p,(62 ) pomc[103120],(7 ) and pomc[124136],(62 ) as shown in table 1 . we also identify novel n - terminal acetylated peptides such as proenkephalin[198209 ] , pomc[124137 ] , secretogranin 2[287297 ] , secretogranin 5[198210 ] , secretogranin 5[180195 ] , the pyro - glu forms of secretogranin 1[597611 ] , proenkephalin b[228248 ] , and neurotensin precursor[150162 ] ( table 1 ) . although our processing steps reduce protein degradation , we did detect several peptides from proteins that are not prohormones ( table 2 ) . mbp is the second most abundant protein in the central nervous system and is present in the peripheral nervous system as well.(55 ) mbp can have different ptms such as phosphorylation of serine and threonine residues , deamidation of asn or gln , citrullination and methylation of arginine residues , and n - terminal acetylation of thr residues . mbp plays an important function in axon myelination , but other possible roles of mbp are hinted , such as significant changes in mrna levels on exposure to cocaine . three peptides derived from mbp are detected in the current study of the son peptidome , one of which exhibits n - terminal acetylation of thr residues ( table 2 ) . our neuropeptidomics study of this small nucleus extends to the single cell level using maldi ms . although in son extracts we are able to identify several fragments derived from provasopressin , our single cell experiments detect the characteristic peptides processed from this hormone . the major drawback of single cell ms is the low amount of material available for analysis ; some peptides are often present below the detection level of the employed ms approach . because of our careful isolation approach , extracellular degradation products from peptides and proteins are not normally detected . thus , our detection of cart - related peptide in a portion of the studied mcn cells is exciting and suggests that we are observing cell - to - cell differences in the peptide complement in a  homogenous  population of mcns . with the ability to perform peptidome studies in well - defined , smaller anatomical regions from the brain slice , coupled with novel methods of characterizing stimulation - dependent peptide release recently reported,(27 ) the ability to link peptide complement to function has become possible .
the mammalian supraoptic nucleus ( son ) is a neuroendocrine center in the brain regulating a variety of physiological functions . within the son , 
 peptidergic magnocellular neurons that project to the neurohypophysis ( posterior pituitary ) are involved in controlling osmotic balance , lactation , and parturition , partly through secretion of signaling peptides such as oxytocin and vasopressin into the blood . 
 an improved understanding of son activity and function requires identification and characterization of the peptides used by the son . here , 
 small - volume sample preparation approaches are optimized for neuropeptidomic studies of isolated son samples ranging from entire nuclei down to single magnocellular neurons . unlike most previous mammalian peptidome studies , tissues are not immediately heated or microwaved . 
 son samples are obtained from ex vivo brain slice preparations via tissue punch and the samples processed through sequential steps of peptide extraction . 
 analyses of the samples via liquid chromatography mass spectrometry and tandem mass spectrometry result in the identification of 85 peptides , including 20 unique peptides from known prohormones . as the sample size 
 is further reduced , the depth of peptide coverage decreases ; however , even from individually isolated magnocellular neuroendocrine cells , vasopressin and several other peptides are detected .