B American Society for Mass Spectrometry, 2015DOI: 10.1007/s13361-015-1251-6

J. Am. Soc. Mass Spectrom. (2015) 26:1970Y1980

FOCUS: MASS SPECTROMETRY-BASED STRATEGIESFOR NEUROPROTEOMICS AND PEPTIDOMICS: RESEARCH ARTICLE

Neuropeptidomics Mass Spectrometry Reveals SignalingNetworks Generated by Distinct ProteasePathways in Human Systems

Vivian Hook,1,2 Nuno Bandeira1,3

1Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093-0719, USA2School of Medicine, Department of Neurosciences and Department of Pharmacology, University of California, San Diego, LaJolla, CA 92093-0719, USA3Department of Computer Science and Engineering, University of California, San Diego, La Jolla, CA 92093-0719, USA

Abstract. Neuropeptides regulate intercellular signaling as neurotransmitters of thecentral and peripheral nervous systems, and as peptide hormones in the endocrinesystem. Diverse neuropeptides of distinct primary sequences of various lengths,often with post-translational modifications, coordinate and integrate regulation ofphysiological functions. Mass spectrometry-based analysis of the diverseneuropeptide structures in neuropeptidomics research is necessary to define the fullcomplement of neuropeptide signaling molecules. Human neuropeptidomics hasnotable importance in defining normal and dysfunctional neuropeptide signaling inhuman health and disease. Neuropeptidomics has great potential for expansion intranslational research opportunities for defining neuropeptide mechanisms of human

diseases, providing novel neuropeptide drug targets for drug discovery, and monitoring neuropeptides asbiomarkers of drug responses. In consideration of the high impact of human neuropeptidomics for health, anobserved gap in this discipline is the few published articles in human neuropeptidomics compared with, forexample, human proteomics and related mass spectrometry disciplines. Focus on human neuropeptidomics willadvance new knowledge of the complex neuropeptide signaling networks participating in the fine control ofneuroendocrine systems. This commentary review article discusses several human neuropeptidomicsaccomplishments that illustrate the rapidly expanding diversity of neuropeptides generated by proteaseprocessing of pro-neuropeptide precursors occurring within the secretory vesicle proteome. Of particular interestis the finding that human-specific cathepsin V participates in producing enkephalin and likely other neuropep-tides, indicating unique proteolytic mechanisms for generating human neuropeptides. The field of humanneuropeptidomics has great promise to solve new mechanisms in disease conditions, leading to new drugtargets and therapeutic agents for human diseases.Keywords: Neuropeptidomics, Neurotransmitters, Peptide hormones, Intercellular signaling, Proteases,Protease cleavage sites, Cathepsin L, Cathepsin V, Proprotein convertase, Proteomics, Secretory vesicles,Bioinformatics, Human signaling networks, Biomarker, Drug response

Received: 30 June 2015/Revised: 30 July 2015/Accepted: 5 August 2015/Published Online: 19 October 2015

Introduction

Neuropeptides mediate intercellular signaling in the ner-vous and endocrine systems in the integrated and coordi-nated control of physiological functions (Figure 1) [1–5]. The

tremendous diversity of the huge spectrum of neuropeptideshighlights their significance in multiple regulatory functions. Inthe nervous system, neuropeptides function as peptide neurotrans-mitters for chemical communication among neural circuits in thebrain and in the peripheral sympathetic and parasympatheticnervous systems. Neuropeptides link communication signalsamong nervous and endocrine systems. In endocrine functions,peptide hormones regulate physiological homeostasis andresponses to environmental stresses that involve all organ systems.

Electronic supplementary material The online version of this article (doi:10.1007/s13361-015-1251-6) contains supplementary material, which is availableto authorized users.

Correspondence to: Vivian Hook; e-mail: vhook@ucsd.edu

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Neuropeptide and Classical Small MoleculeNeurotransmitters in the Nervous System

Neuropeptides function as peptide neurotransmitters alongwith the classical small molecule neurotransmitters, the twomain categories of neurotransmitters in the nervous system.Neuropeptides are represented by diverse peptide sequencestypically consisting of about 3-40 amino acid residues, andmany contain post-translational modifications. It is estimatedthat hundreds to thousands of different neuropeptides are uti-lized in numerous organisms, with many yet to be discovered.The neuropeptides regulate pain, appetite, cognition, and mi-graine via the endorphin, NPY, galanin, and CGRP peptides,respectively, as examples. In contrast to neuropeptides, theclassical neurotransmitters consist of small molecules that are

largely generated by modifications of single amino acids, suchas norepinephrine synthesized from tyrosine and serotoninsynthesized from tryptophan. Classical transmitters such asacetylcholine are synthesized by enzymatic reactions, in thiscase, from choline and acetyl-CoA by choline acetyl transfer-ase. The Bneuropeptide^ and Bclassical^ neurotransmitters to-gether mediate intercellular signaling in the nervous systemamong neurons, as well as glia cells [1–5].

The distinct primary sequences of neuropeptides define theirselective and potent biological actions, mediated in large partby G-protein coupled receptors. A given neuropeptide mayfunction in both the nervous and endocrine systems (Table 1).For example, adrenocorticotropin hormone (ACTH) is aneuromodulator in brain, and also regulates peripheral

Figure 1. Neuropeptides for neuronal and endocrine cell-cell communication. (a) Neuropeptides in the central nervous system ofbrain. Brain neuropeptides function as peptide neurotransmitters to mediate chemical cell–cell communications among neurons.Neuropeptides are synthesized within secretory vesicles that are transported from the neuronal cell body via the axon to nerveterminals. The pro-neuropeptide (or prohormone) is packaged with newly formed secretory vesicles in the cell body, and proteolyticprocessing of the precursor protein occurs during axonal transport and maturation of the secretory vesicle. Matureprocessed neuropeptides are contained within secretory vesicles at the synapse where activity-dependent, regulated secretion ofneuropeptides occurs to mediate neurotransmission via neuropeptide activation of peptidergic receptors. (b) Neuropeptides in theperipheral nervous system and endocrine systems for regulation of physiological organ functions. The peripheral nervous systemregulates all organ systems, linking the central nervous system of the brain with peripheral neuronal control of physiologicalfunctions. In the body, neuropeptides also function as hormones that mediate endocrine cell-cell communication

V. Hook and N. Bandeira: Neuropeptidomics Illustrates Diverse Neuropeptides 1971

glucocorticoid metabolism controlled by the pituitary and ad-renal glands. Enkephalin neuropeptides function as neurotrans-mitters in the brain in the regulation of pain, stress anxiety,depression, drug reward and addiction, cardiovascular control,intestinal motility, and immune functions [6, 7]. The neuropep-tides β-endorphin, neuropeptide Y (NPY), galanin, corticotropin-releasing factor, vasopressin, insulin, and numerous others con-trol prominent physiological functions of pain, feeding behaviorand blood pressure regulation, cognition, stress, water balance,and glucose metabolism, respectively. The plethora of physio-logical systems regulated by neuropeptides thus illustrates thatneuropeptides participate in the control of all organ systems.Furthermore, neuropeptides function across different organismsincluding invertebrate, mammals, and human.

Advantages of Neuropeptidomics Analysisof Neuropeptide Structures by Mass Spectrometry

Clearly, the diversity of neuropeptide functions indicates theessential requirement to understand their peptide sequencestructures, including post-translational modifications that de-fine molecular mechanisms of their biological actions. Thedevelopment of mass spectrometry analysis of peptides hasprovided tremendous new knowledge of the peptide structuresof neuropeptides. Even the early discovery of the thyrotropinreleasing hormone (TRH) peptide hormone by RogerGuillemin and Andrew Schally, Nobel prize recipients in1977, utilized mass spectrometry to elucidate the amino acidsequence of thryotropin releasing hormone [8, 9], among thefirst identified hypothalamic releasing hormones. At that time,discovery of the radioimmunoassay (RIA) method by Yalowand Berson in 1960 [10] allowed sensitive measurements ofinsulin and neuropeptides through the subsequent decades, andwas recognized with award of the Nobel prize to RosalynYalow in 1977. Subsequent advances in high resolution massspectrometry analysis of neuropeptides allows unbiased,

objective definition of peptide amino acid sequence structuresthat are not possible with antibody-based neuropeptide detec-tion methods including ELISA and RIA immunoassays.

The defined mass of each neuropeptidemolecule predicts itsamino acid sequence structure with high accuracy. Moreover,the combination of liquid chromatography coupled with massspectrometry (LC-MS/MS) enables analyses of hundreds, andnearly thousands, of neuropeptide structures in single experi-ments. With the rapid acquisition of large LC-MS/MS datasetsof neuropeptides, development of neuropeptide-focused bioin-formatics strategies are required for appropriate identification.The high throughput LC-MS/MS and bioinformatics analysesof peptides of the nervous and endocrine systems are termedBneuropeptidomics.^ The term Bneuropeptidomics^ refers toglobal identification and quantitation of peptide profiles inneuroendocrine systems by LC-MS/MS technologies.

Neuropeptidomics allows systems analyses of intercellularsignaling networks in the regulation of nervous and endocrinecontrol, especially in human functions of physiological homeo-stasis, human disease conditions, and responses to changesenvironmental conditions including drug therapeutics.Investigation of neuropeptidomics signatures in human diseaseallows opportunities for advancing mechanistic knowledge ofdisease progression, biomarkers for disease states, and newpeptide targets for drug discovery and development. The pro-found importance of neuropeptide regulation of human condi-tions provides the rationale for rapid advances inneuropeptidomics investigation in human health and disease.

Human Neuropeptidomics: Current Statusin the Field

Although the significance of neuropeptidomics regulation ofhuman physiology is undisputed, a search of the literatureindicates the current paucity of neuropeptidomics researchcompared with proteomics mass spectrometry to gain proteinstructural information. PubMed searches of Bpeptidomics^ indi-cates 366 published articles, and search of Bneuropeptidomics^yields 32 articles; but search of Bhuman neuropeptidomics^indicates only nine articles published (Table 2). In contrast, theproteomics field shows substantially greater research activity.BProteomics^ studies number 55,388 articles to date (PubMed),and Bhuman proteomics^ is described so far in 30,141 articles(Table 2). Search of Bpeptide mass spectrometry^ covers bothproteomics and peptidomics since proteomics is conducted byanalysis of tryptic peptide digests, and is illustrated by 50,069articles published to date. The few articles published on Bhumanneuropeptidomics^ clearly demonstrate the high need for expan-sion of the neuropeptidomics field to gain new understanding ofneuropeptide regulation of human biological systems.

For this reason, this review focuses on Bhumanneuropeptidomics^ to illustrate strategies for expansion andacceleration of human neuropeptidomics research. A spectrumof related studies is reviewed here covering human neuropep-tide diversity, human protease pathways for neuropeptide bio-synthesis, secretory vesicle proteomics systems that are utilized

Table 1. Neuropeptides in the Nervous and Endocrine Systems

Neuropeptides Physiological functions

Enkephalins Analgesia, pain reliefβ-Endorphin Analgesia, pain reliefDynorphin Analgesia, pain reliefCRH Stress, glucocorticoid productionACTH Steroid productionα-MSH Skin pigmentation, appetiteInsulin Glucose metabolismGlucagon Glucose metabolismGalanin CognitionNPY Obesity, blood pressureSomatostatin Growth regulationVasopressin Water balanceCalcitonin Calcium regulation, migraineCholecystokinin Learning, memory, appetite

Neuropeptides function as peptide neurotransmitters and peptide hormones.Examples of several neuropeptides and their biological functions are shown inthis table.ACTH = adrencorticotropin hormone; α-MSH = α-melanocyte stimulatinghormone; NPY = neuropeptide Y; CRH = corticotropin releasing hormone.

1972 V. Hook and N. Bandeira: Neuropeptidomics Illustrates Diverse Neuropeptides

for neuropeptide production, and networks of secreted neuro-peptide profiles that undergo coordinate regulation in physio-logical responses. These studies raise important questions to beinvestigated in future studies. Development of NeuroPedia, aneuropeptide database and spectral library, facilitates bioinfor-matics analyses of neuropeptidomics data [11]. Humanneuropeptidomics research has great promise to solve newmechanisms in human neurobiology that can lead to new drugtargets and therapeutic treatments for human diseases. It is,therefore, predicted that human neuropeptidomics is becomingan expanding field of broad interest to biomedical sciences.

Diversity of Human Neuropeptide Pro-files Revealed by Neuropeptidomics:Explosion of Intercellular SignalingPeptides to be DiscoveredThe diversity of human neuropeptides is tremendous andwill expand with neuropeptidomics analyses of the pep-tide sequence structures identified in the central andperipheral nervous system, and the endocrine systems.Neuropeptidomics identification of known and currentneuropeptides utilizes knowledge of the pro-neuro-peptide precursor proteins that undergo proteolyticprocessing to generate neuropeptides. Neuropeptidomicsexperiments will identify numerous peptides, and thequestion will arise about their functions in biologicalprocesses. Such questions will stimulate novel neuropep-tide research.

Pro-Neuropeptide Precursors of ActiveNeuropeptides

Active neuropeptides are generated from inactive pro-neuropeptide precursor proteins, also known as prohormonesfor those in the endocrine systems. A pro-neuropeptide

precursor may contain one copy of the active neuropeptide asrepresented by pro-neuropeptides for NPY, galanin, and vaso-active intestinal polypeptide (VIP) (Supplemental Figure S1)[5]. Or, a pro-neuropeptide may contain several related copiesof the neuropeptide; for example, proenkephalin (PE) containsfour copies of (Met)enkephalin, one copy of the related(Leu)enkephalin, and one copy each of the neuropeptidesME-Arg-Gly-Leu and ME-Arg-Phe (SupplementalFigure S1). Further, a pro-neuropeptide may contain severaldifferent neuropeptides with each having distinct biologicalactions; for example, the proopiomelanocortin precursor con-tains β-endorphin, adrenocorticotropin hormone (ACTH), andmelanocyte stimulating hormone (α-MSH). Of particular inter-est is the tissue-specific expression of the proopiomelanocortin(POMC)-derived neuropeptides.

Although the primary sequences of pro-neuropeptides dif-fer, as the distinct sequences of neuropeptides are the basis fortheir specific biological actions, the neuropeptides are com-monly flanked by dibasic residues at their NH2- and COOH-termini within the pro-neuropeptide (Supplemental Figure S1).The dibasic residues Lys-Arg (KR) most often flank the neu-ropeptides, and the dibasic sites Lys-Lys, Arg-Arg, and Arg-Lys also occur. These paired basic residues represent sites ofproteolytic processing to liberate neuropeptides from their pre-cursor proteins. Processing may also occur at monobasic Argsites as well as at monobasic residue sites (as in POMC).Processing at nonbasic residues may occur but is not as welldefined as processing at dibasic residues of pro-neuropeptides.

Intervening Peptide SequencesWithin Pro-Neuropeptides

It is of interest that analyses of pro-neuropeptide sequencesindicate that major portions of the pro-neuropeptides haveunknown function. Known active neuropeptides have beenthe subject of peptide neurotransmission through synthesis,secretion, and activation of specific receptors However, thereare many intervening peptide sequences present betweenknown active neuropeptides within the precursors, but littleattention has been given to investigating such interveningsequences. Neuropeptidomics is an ideal strategy to answerthe question, What peptide products are generated from pro-neuropeptides? Neuropeptidomics may reveal new andexisting neuropeptides.

Neuropeptidomics Reveals that Pro-Neuropeptidesare Converted into Intact Intervening Sequencesand Known Active Neuropeptides

Neuropeptidomics analysis by nano-liquid chromatographytandem mass spectrometry (LC-MS/MS) of human secretoryvesicles (isolated from human pheochromocytoma) is capableof revealing a repertoire of processed peptides derived from asingle pro-neuropeptide [12]. Furthermore, LC-MS/MS in oneexperiment provides data for the spectrum of peptide productsderived from multiple pro-neuropeptides. These experiments

Table 2. Neuropeptidomics Compared with Proteomics Publications

Topic Number of citations(PubMed)

Dates ofpublications

Neuropeptidomics:Human neuropeptidomics 9 2006–2015Neuropeptidomics 32 2004–2015Peptidomics 366 2001–2015

Proteomics:Human proteomics 30,141 1998–2015Proteomics 55,388 1998–2015

Search of PubMed was conducted using the key words listed above. Thenumber of publications shown for each search (as of July, 2015) are shown. Itis realized that use of related search phrases or terms will also reveal publishedarticles on these topics; however, the recent terminology of Bneuropeptidomics^and Bpeptidomics^ in the literature is shown to occur since approximately theyear 2001. A tremendous difference is observed in the number of articlespublished for Bhuman neuropeptidomics^ compared with Bhumanproteomics.^ Among the Bneuropeptidomics^ and Bpeptidomics^ topics,Bhuman neuropeptidomics^ is a small portion of such published studies to date.

V. Hook and N. Bandeira: Neuropeptidomics Illustrates Diverse Neuropeptides 1973

were conducted with a low molecular weight (MW) pool(obtained by a 10kDa Millipore filtration membrane).

Neuropeptidomics illustrated the multiple peptide productsin human pheochromocytoma secretory vesicles derived fromproenkephalin (PE), pro-NPY, pro-SAAS, and thechromogranins A, B, and C (CgA, CgB, CgC, respectively)[12]. Features of peptides derived from PE and CgA arehighlighted.

Neuropeptidomics revealed numerous extended forms of(Met)enkephalin that included Bintervening^ sequences ofnon-enkephalin domains of proenkephalin (Figure 2).Furthermore, intervening peptide sequences that do not includeenkephalin were identified. Some intervening peptide domainswere not detected (residues 145-161). Significantly, the pres-ence of such intervening peptide sequences has not been ob-served in prior studies.

These neuropeptidomics findings identified numerousCgA-derived peptide domains of catestatin, vasostatin,parastatin, and related neuropeptides derived from CgA(Supplemental Figure S2). Several intervening peptidesderived from CgA were identified. The low MW pooldid not include all intervening peptides, which suggeststhat their presence as large intermediate polypeptidesgreater than 10kDa that are derived from the CgA pre-cursor of ~68kDa.

The presence of intact intervening peptide sequences andknown active neuropeptides derived from pro-neuropeptideprecursors indicates that an explosion of new neuropeptideswill be revealed by neuropeptidomics. The question of possiblebiological functions of the intervening peptides will be ofinterest to address in future research. Neuropeptidomics iskey for unbiased analyses of all peptides derived from precur-sor proteins, in contrast to traditional focused immunoassaysthat eachmeasure only one pre-selected neuropeptide. Findingsin the field have, thus far, observed only the Btip of the iceberg^of the full spectrum of neuropeptides. It will be exciting to gainunderstanding of the full spectrum of neuropeptides derivedfrom pro-neuropeptide precursors, and define their biologicalfunctions.

Protease Cleavage Sites of Pro-NeuropeptidesObserved by Neuropeptidomics Data: DibasicResidue Processing and Novel Cleavage Sitesof Pro-Neuropeptide Processing

Neuropeptidomics of human secretory vesicles (human adrenalmedullary pheochromocytoma) has defined proteolytic peptideproducts of several pro-neuropeptides, includingproenkephalin, pro-NPY (neuropeptide Y), pro-SAAS (Ser-Ala-Ala-Ser related peptides), chromogranin A, chromograninB, and secretogranin II (SCG2) that has also been known aschromogranin C [12]. Evaluation of the adjacent peptide se-quences at the N- and C-termini of identified peptides revealedthat these peptides were derived from processing at classicaldibasic residue sites of pro-neuropeptides, and also at novelcleavage sites.

Classical dibasic residue cleavage site motifs flanking theN- and C-termini of endogenous peptides were prevalent,shown by LOGO maps, which illustrated processing at KR,KK, RK, and RR sites (Figure 3a). These data are consistentwith pro-neuropeptide processing by proteases known to pos-sess cleavage specificities for dibasic residue sites. These pro-teases consist of the subtilisin-like proprotein convertases andcysteine cathepsin protease pathways (described in the sectionon human protease pathways).

Novel cleavage sites of pro-neuropeptides were also identi-fied by neuropeptidomics data and illustrated by LOGO maps(Figure 3b). There analyses found an abundance of acidicamino acids (E, glutamate) at the P1 position of putativecleavage sites (cleavage site is P1-↓P1′). These cleavage sitesoccur at the junctions of known active neuropeptides andintervening sequences of pro-neuropeptides. Thus far, prote-ases cleaving at glutamate residues for neuropeptide productionhave not yet been identified.

Based on searches of the literature to date, the study byGupta et al., 2010 [12] is among the first to report on humanpro-neuropeptide cleavage sites via neuropeptidomics. Futurehuman neuropeptidomics research will benefit from compre-hensive studies of endogenous peptides among human neuro-endocrine tissues that will likely define new and existing neu-ropeptides that are generated by novel and classical proteolyticprocessing of human pro-neuropeptides.

Bioinformatics for Neuropeptidomicsby NeuroPedia: Neuropeptide Data-base and Spectral LibraryBioinformatics addressing the particular features ofneuropeptidomics data is necessary to facilitate high qualitydata analyses. Global analysis of neuropeptidomics expressiondata is necessary for understanding the role and regulation ofneuropeptide forms in health, disease, and drug treatments.However, the unique properties of neuropeptides, with respectto very short and very long nontryptic peptide sequences,presents difficulties for identification from tandem mass spec-trometry data (MS/MS) with popular database search tools(such as SEQUEST or Mascot) [13, 14]. Short neuropeptidescan lead to inaccurate search results because the databasesearch tools usually assign lower scores to short peptides.Alternatively, long or nontryptic neuropeptides are difficult toidentify because most database search tools are trained fortryptic peptides cleaved at K/R and because peptide fragmen-tation processes for long neuropeptides is usually inefficient.Further, searching larger databases takes more time because ofthe number of comparisons, and reduces the number identifi-cations with the caveat of greater choices for false positives[15]. Thus, while some neuropeptides can be identified withcurrent bioinformatics approaches, complete neuropeptidomicsrequires novel computational tools for identifying both shortand long neuropeptides by MS/MS [11].

1974 V. Hook and N. Bandeira: Neuropeptidomics Illustrates Diverse Neuropeptides

For these reasons, NeuroPedia is being developed as a spe-cialized neuropeptide database and spectral library that is direct-ly searchable using mass spectrometry data [11]. NeuroPediaimproves sensitivity by targeted searching of a small neuropep-tide sequence database, and provides enhanced identificationefficiency, sensitivity, and reliability. NeuroPedia spectral librar-ies are compatible with the publicly available spectral librarysearch tool M-SPLIT [16] and can integrate with other spectrallibrary formats. NeuroPedia provides annotated spectrum im-ages for every library spectrum and separates spectral librariesby species, enzyme digestion, and MS instrument.

The NeuroPedia spectral library contains a total of 3401identified spectra in MGF files. The NeuroPedia sequencedatabase contains 847 neuropeptides from human, chimpanzee,mouse, rat, cow, sea hare, rhesus macaque, and leech. UsingInsPecT or any other database search tool, new MS/MS datacan be searched against this sequence database. NeuroPediaoffers the advantages of being rapid and precise in the identi-fication of small or nontryptic neuropeptides.

Comparison of NeuroPedia with the online neuropeptiderepository (at www.neuropeptides.nl) shows that this resourceis not designed for peptide identification from MS/MS data

[17]. This repository provides non-searchable neuropeptidesequences, gene names, precursor names, and expected humanbrain expression. Users must search their data using otherpeptide database search tools and compare the results againstthe neuropeptide list. This process is less sensitive and utilizestime-consumingmanual matching of searches to information incurrent resources.

The www.neuropeptides.nl are complemented by recentdevelopment of NeuroPep [18], which is a comprehensiveresource of neuropeptides, which holds 5949 non-redundant neu-ropeptide entries from 493 vertebrate and invertebrate organisms.Each peptide entry of the database contains organisms, tissues,families, names, modifications, 3D structures (if known), andliterature references. It is noted that NeuroPep and www.neuropeptides.nl are valuable for providing information aboutneuropeptides, but they do not cover computational strategiesfor neuropeptide identification as addressed by NeuroPedia.

Recently, accurate assignment of significance to neuropep-tide identifications was developed using Monte Carlo k-permuted decoy databases [19]. The straightforward MonteCarlo permutation testing can be combined with existing pep-tide identification software for accurate neuropeptide detection.

Neuropeptidomics of Proenkephalin-Derived Peptides1 10 20 30 40 50 60E C S Q D C A T C S Y R L V R P A D I N F L A C V M E C E G K L P S L K I W E T C K E L L Q L S K P E L P Q D G T S T Lprosegment -

61 70 80 90 100 110 120R E N S K P E E S H L L A K R Y G G F M K R Y G G F M K K M D E L Y P M E P E E E A N G S E I L A K R Y G G F M K K D A

- prosegment, cont. Met-enk Met-enkephalin Met-enkephalin__

121 130 140 150 160 170 180E E D D S L A N S S D L L K E L L E T G D N R E R S H H Q D G S D N E E E V S K R Y G G F M R G L K R S P Q L E D E A K

Octapeptide_ __ _____________

181 190 200 210 220 230 240 243E L Q K R Y G G F M R R V G R P E W W M D Y Q K R Y G G F L K R F A E A L P S D E E G E S Y S K E V P E M E K R Y G G F M R F

Met-enk Leu-enkephalin Heptapeptide_____________

*

QTOF-Ins-no enzyme Trap-Ins-noenzyme basic residuesQTOF-SM-noEnz Trap-SM-noEnz cleavage sites

indicated domain

_

Figure 2. Human proenkephalin-derived neuropeptides assessed by neuropeptidomics. Neuropeptidomic studies investigatedendogenous peptides derived from human proenkephalin in secretory vesicles of human pheochromocytoma [12]. Neuropeptidesderived from human proenkephalin (PE) are illustrated with respect to their location within PE. Peptides were identified by ion-trapandQTOFMS/MS, combinedwith InsPecT (Ins) andSpectrumMill (SM) bioinformatics analyses ofMS/MSdata at 1%FDR (with theexception of Leu-enkephalin that was indicated at 5% FDR). Peptides identified under each of these conditions weremapped to PE,illustrated by colored lines: QTOF MS/MS data analyzed by InsPect (Ins, orange) or Spectrum Mill (SM, yellow), and ion-trap (Trap)analyzed by insPect (Ins, green) or SM (olive). Within PE, the active enkephalin neuropeptides sequences are shown in yellow.Dibasic cleavage sites are highlighted by boxes; in addition, monobasic residues within PE are shown. (Hyphens at the end of somelines indicate peptides that were split between two lines)

V. Hook and N. Bandeira: Neuropeptidomics Illustrates Diverse Neuropeptides 1975

http://www.neuropeptides.nl/http://www.neuropeptides.nl/http://www.neuropeptides.nl/http://www.neuropeptides.nl/

NeuroPedia uniquely utilizes neuropeptide spectral librariesto enhance neuropeptide identification by MS/MS methods.

Expansion of NeuroPedia involving neuropeptidomics investiga-tors will significantly advance human neuropeptidomics knowl-edge, as well as neuropeptidomics in numerous organisms.Because the breadth of the diversity of neuropeptides has yet tobe fully understood, continued development of NeuroPedia willbe instrumental in defining the full spectrum of neuropeptidestructures that regulate biological systems in humans and allorganisms. Further, arrangement of NeuroPedia for integrationwith other ongoing neuropeptide and peptide bioinformaticssystems for MS/MS identification will benefit the field for com-prehensive analysis of human neuropeptidomics.

Human-Specific Protease Pathwaysfor Neuropeptide BiosynthesisProteases for processing pro-neuropeptides are required for theproduction of biologically active neuropeptides from inactiveprecursor proteins. Two distinct protease pathways forsubtilisin-like proprotein convertases and cysteine cathepsinproteases have been demonstrated to participate in neuropep-tide biosynthesis (Figure 4). Notably, emerging evidence illus-trates the human-specific cathepsin V of the cysteine protease

Figure 3. Neuropeptidomics reveals processing of pro-neuropeptides at dibasic residue sites at and non-dibasic resi-dues. (a) Dibasic residue cleavage sites. Dibasic residue sites aremajor proteolytic cleavage sites of pro-neuropeptides in humansecretory vesicles. Sequence LOGOmaps of theN- andC-terminiof neuropeptides in human secretory vesicles were generated byevaluation of adjacent residues to the identified peptides with theirpro-neuropeptide precursors, as we reported [12]. (i) N-terminalcleavage sites of identified neuropeptides. The N-termini of peptidesare indicated by the arrow, with illustration of the flanking aminoacid sequences present within the peptides’ pro-neuropeptides.The x-axis shows residues at the P1-P15 positions relative to thecleavage sties at P1-↓P1′ residues. The relative frequency ofamino acids at each position is illustrated (y-axis). (ii) C-terminalcleavage sites of identified neuropeptides. The C-termini of peptidesare indicated by the arrow, with residues of respective pro-neuropeptides flanking the C-termini at P1′-P15′ positions. (b)Novel non-dibasic residue cleavage sites. Non-dibasic residuecleavage sites of pro-neuropeptides are illustrated after removalof the dibasic sites observed in the data. (i) N-terminal cleavage sites.P1-P15 residues flanking theN-termini of identifiedpeptideswithintheir pro-neuropeptides are illustrated. (ii) C-terminal cleavage sites.P1′-P15′ residues flanking the C-termini of identified peptideswithin their pro-neuropeptide precursors are illustrated

Figure 4. Human cysteine and serine protease pathways forneuropeptide production. Distinct cysteine protease andsubtilisin-like protease pathways participate in pro-neuropeptide processing [5]. The cysteine proteases cathepsinL and human-specific cathepsin V in secretory vesicles functionsas a processing enzyme for the production of neuropeptides.Human cathepsin L and cathepsin V cleave at dibasic residuesites within the pro-neuropeptide. Subsequent to cathepsins Land V, an aminopeptidase is needed to remove N-terminal basicresidues, and carboxypeptidase E (CPE) removes C-terminalbasic residues to generate active neuropeptides. The subtilisin-like protease pathway involves the proprotein convertasesPC1/3 and PC2. The PC enzymes preferentially cleave at theC-terminal side of dibasic processing sites, which results in pep-tide intermediates with basic residue extensions at their C-terminithat are removed by carboxypeptidase E. In mammalian speciesother than human, cathepsin L with the PC1/3 and PC2 prote-ases participate in neuropeptide production

1976 V. Hook and N. Bandeira: Neuropeptidomics Illustrates Diverse Neuropeptides

pathway as a human-specific protease for production of neuro-peptides in human biological systems [20]. This exciting find-ing implicates human-selective approaches for regulating neu-ropeptide production in physiological systems.

Distinct Protease Pathways for NeuropeptideBiosynthesis

The two protease pathways for processing at dibasic residueswere identified through different approaches of (1) gene ho-mology cloning of mammalian protease genes based on ho-mology to the yeast kex 2 gene that generates α-mating factorpeptide hormone, and (2) activity-based mass spectrometryidentification of proenkephalin and pro-neuropeptide process-ing proteases in mammalian systems. These approaches iden-tified distinct protease pathways for pro-neuropeptide process-ing consisting of (1) the subtilisin-like proprotein convertases(PC1/3 and PC2) protease pathway [5, 21], and the (2) cysteinecathepsin L and cathepsin V protease pathway (Figure 4) [5,20, 22]. Proteases of both pathways cleave primarily at dibasicresidue cleavage sites within pro-neuropeptides.

PC1/3 and PC2 family members participate in neuroendo-crine production of neuropeptides, demonstrated through geneknockou t and gene express ion s tud ie s , and byneuropeptidomics characterization in mice with knockout of

the PC1/3 or PC2 genes [5, 21–24]. Because of the limitedscope of this review article, readers are referred to the extensiveliterature on PC1/3 and PC2 [5, 21–27] that indicate the currentknowledge of PC1/3 and PC2 in neuropeptide production.

Human-Specific Cathepsin V and Human CathepsinL of the Cysteine Protease Pathwayfor Neuropeptide Production

The neuropeptide cysteine protease pathway was elucidated byidentifying major pro-neuropeptide cleaving activity, using

Protein Interaction Network of Human Neuropeptide Secretory Vesicles

Soluble Protein Membrane Protein

Figure 5. Proteomics and systems biology of human neuro-peptide secretory vesicles. Proteomics data of humanneuropeptide-containing secretory vesicles (dense core secre-tory vesicle, DCSV, type) was subjected to Cytoscape systemsbiology analyses for predicting protein interaction networks [31,32]. The functional protein categories are shown on the righthand side. Based on quantitative NASF data of the MS/MSidentified proteins, proteins are indicated as soluble (greencircles) or membrane (red circles) proteins, or present in bothsoluble andmembrane at similar levels (yellow circles). Proteinsshown as gray circles were identified, but not quantitated sincethey did not meet the criteria for quantitation of at least threeidentifications out of four nano-LC-MS/MS runs

Figure 6. Vasoactive neuropeptides regulated by ACE inhibi-tor drug therapeutics. The effects of an ACE (angiotensinconverting enzyme) inhibitor, captopril, on levels of plasmavasoactive peptides were analyzed in time-course studies bynano-LC-MS/MSwith quantitation using stable isotope-labeledinternal peptide standards [36]. ACE inhibitors are utilized asanti-hypertensive drugs. Chromatographic separation of targetpeptides and multiple reaction monitoring (MRM) providedquantitation of angiotensin I (Ang I), Ang II, Ang1-7, bradykinin1-8 (BK 1-8), BK-2-9, and kallidin (KD). Results show significantreduction by the ACE inhibitor of the angiotensin peptides, withan interesting concomitant increase in plasma bradykinins andkallidin (potent vasodilators). The percent change in plasmaconcentration at different times after drug administration isshown in the table below the bar graph. Results illustrate theutility of simultaneous profiling of multiple peptides using massspectrometry analysis to monitor drug-induced changes in va-soactive neuropeptides

V. Hook and N. Bandeira: Neuropeptidomics Illustrates Diverse Neuropeptides 1977

recombinant proenkephalin (PE) as substrate, in secretory ves-icles where pro-neuropeptide processing occurs [5]. Chemicallabeling of the PE-cleaving activity with the activity-basedprobe DCG04 for cysteine proteases allowed identification of theenzyme by mass spectrometry [28], indicating cathepsin L as theprocessing protease. Cathepsin L gene knockout in mice con-firmed its major role in the production of multiple neuropeptides,including ACTH, α-MSH, β-endorphin, CCK (cholescystokinin),NPY, and others [5, 22]. Furthermore, the colocalization ofcathepsin L with neuropeptides in secretory vesicles indicates thesecretory vesicle as a new organelle location for the biologicalfunction of cathepsin L in producing bioactive peptides.

Notably, the human genome possesses highly homologoushuman cathepsin L and human cathepsin V proteases [20, 29,30]. No orthologues of human cathepsin V is present in mouseand other mammalian species [29, 30], indicating that cathep-sin V is a human-specific protease gene. Mouse cathepsin Lpossesses high homology with human cathepsin V (74.6%homology in protein sequence), which is greater than theexcellent homology of human cathepsin L and mouse cathep-sin L (71.5% homology in protein sequence) (SupplementalFigure S3a). These homologies predict that human-specificcathepsin V can participate in neuropeptide production.

Indeed, human cathepsin V participates in generating en-kephalin neuropeptide from its proenkephalin (PE) precursor[20]. Gene silencing of cathepsin V (by siRNA) substantiallyreduced production of enkephalin by more than 80%(Supplemental Figure S3b). Further, cathepsin V cleaves PEat dibasic residue sites to generate PE-derived intermediatesand (Met)enkephalin. Cathepsin V is present in secretory ves-icles with enkephalin, and cathepsin V is present human brainregions that produce enkephalin neuropeptides. These datademonstrate the significant role of human-specific cathepsinV in enkephalin neuropeptide production. It will be importantto conduct human neuropeptidomics studies to gain under-standing of human neuropeptides that are produced by humancathepsin V and cathepsin L. Human-focused studies willfurther define the roles of the cysteine protease pathway ofcathepsin V and cathepsin L with the subtilisin-like proteasepathway of PC1/3 and PC2 convertases.

Human Secretory Vesicle Proteomefor Neuropeptide Biosynthesis, Stor-age, and SecretionNeuropeptide production occurs largely in secretory vesicleswhich then store and secrete neuropeptides for intercellularsignaling. The pro-neuropeptides and the processing proteasesfunction within the environment of the secretory vesicle prote-ome. In neurons, pro-neuropeptide processing occurs withinsecretory vesicles during their transport along the axon to nerveterminals for neuropeptide release as neurotransmitters.Proteomic studies of human neuropeptide-containing secretoryvesicles can identify the functional protein categories utilizedfor neuropeptide production and secretion.

The protein architecture of human dense core secretoryvesicles (DCSV) that produce neuropeptides was subjected toquantitative proteomics and systems biology analysis, usinghuman DCSV purified from human pheochromocytoma [31].Over 600 human DCSV proteins were identified with quanti-tation of over 300 proteins, revealing that most proteins partic-ipate in producing neurotransmitters and neurohumoral factorscons is t ing of pro-neuropept ides , pro teases , andneurotransmitter/neurohumoral factor proteins. Proteins thatregulate the internal secretory vesicle conditions includedATPases, chaperones, and those that regulate reduction-oxidation conditions. Protein functions in biochemical, secre-tory, and morphologic functions of secretory vesicles are alsopresent.

Organization of human secretory vesicle proteomics data ina systems biology format provided knowledge of soluble andmembrane proteins of the organelle (Figure 5). Protein inter-action networks were analyzed by Cytoscape, a platform forcomplex network analysis and visualization [31, 32].Construction of potential DCSV protein interaction networkswas achieved by query of proteins reported to display protein–protein interactions in the Michigan Molecular Interaction(MiMI) database [31]. The human DCSV network map repre-sents a model of the protein architecture of this organelle(Figure 5). These protein systems support the function ofsecretory vesicles for production and secretion ofneuropeptides.

Modeling Neuropeptidomicsin Human-Induced Pluripotent StemCells Differentiated into NeuronsNeuronal models of human neuropeptide producing cells willenhance investigation of the human proteases responsible forproducing profiles of neuropeptides in neuropeptidomics stud-ies. Investigation of protease pathways utilized in the humannervous system of the brain and peripheral sympathetic andparasympathetic systems is necessary to gain knowledge ofhuman neuropeptidomics biosynthesis. Human patient-derived human-induced pluripotent cells (hiPSC) differentiatedinto neurons as well as glia provide an ideal strategy to definethe human neurobiology of neuropeptide systems.

The first question to address about hiPSC neurons is theirability to synthesize and secrete neuropeptides. Recent studiesshow that hiPSC neurons produce enkephalin, dynorphin, andother neuropeptides that are secreted in an activity-dependentmanner [33]. Moreover, the classical catecholamine neuro-transmitters are also produced and secreted, indicating thathiPSC neurons model human neurotransmitter productionand secretion. These seminal findings show that hiPSC neuronsderived from patients provide human models of the humanneurobiology of neuropeptides in health and disease.

Model hiPSC neurons from patients with neurologic, neu-rodegeneration, mental disorders, and brain dysfunctions[33–35] provide windows into human neuropeptide

1978 V. Hook and N. Bandeira: Neuropeptidomics Illustrates Diverse Neuropeptides

functions. Neuropeptidomics may reveal new mechanismsof human disease conditions. These so-called Bdisease-in-a-dish^ studies are revolutionizing mechanistic studies ofhuman disease conditions and will underscore thesignificance of neuropeptidomics systems in humanhealth and disease.

Human Neuropeptidomics to DefineNeuropeptide Networks in Healthand DiseaseRelated neuropeptides function together in the regulation ofparticular physiological processes. For example, regulation ofblood pressure is achieved by the vasoactive neuropeptidesangiotensin, bradykinin, vasopressin, and others. Thecontrol of pain relief is regulated by the endogenous opioidneuropeptides composed of the related enkephalin, β-endor-phin, dynorphin, and nociceptin neuropeptides [6, 7].Neuropeptidomics allows evaluation of the dynamic changesin neuropeptide profiles that regulate physiological functions.Furthermore, neuropeptidomics provides knowledge of howthey are regulated in response to therapeutic drug agents usedto treat human disease conditions.

For example, the angiotensin converting enzyme (ACE)inhibitor, captopril, is utilized as an anti-hypertensive drug.ACE converts angiotensin I to angiotensin II, which is a vaso-constrictor and elevates blood pressure. Captopril inhibits ACEto result in reduced blood pressure. While captopril blocks theformation of angiotensin II, the drug regulates multiple vaso-active neuropeptides as demonstrated by neuropeptidomicsstudies [36]. Quantitative neuropeptidomics with MRM dem-onstrated that captopril not only decreases plasma angiotensinII but also regulates bradykinin and kallidin in a temporalmanner (Figure 6, for rats administered captopril). Future ex-tension of these strategies to human plasma neuropeptidomicswill be valuable to define how changes in networks of neuro-peptides participate in the regulation of physiologicalprocesses.

Future human neuropeptidomics research will be significantfor gaining new mechanistic understanding of human disease,providing new neuropeptide system components as drug tar-gets for discovery of new therapeutic agents, and monitoringdisease and therapeutic responses by neuropeptide biomarkersdefined via neuropeptidomics investigation. Humanneuropeptidomics research is predicted to undergo remarkableexpansion and contribute to improving human health.

AcknowledgmentsThis work was supported by grants from the National Institutesof Heal th (NIH) (R01DA04271, R01MH077305,P01HL58120), a NARSAD Distinguished Investigator grant,and an Academic Senate Award from UC San Diego to V.H.,and by NIH P41 GM103484 to N.B. and P.P. Contributions byco-authors of cited work is acknowledged: Dr. Steven Bark

(Assistant Professor, University of Houston), Dr. Nitin Gupta(Assistant Professor, Indian Institute of Technology), Dr.Daniel O’Connor (Professor, University of California, SanDiego), Dr. Pavel Pevzner (Professor, University of California,San Diego), Dr. Jill Wegrzyn (Assistant Professor, Universityof Connecticut).

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Pathways in Human SystemsAbstractSection12Section23Section24Section25

Section16Section27Section28Section29Section210

Section111Section112Section213Section214

Section115Section116Section117AcknowledgmentsReferences