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Journal of Neuroscience Methods 197 (2011) 231–237

Contents lists available at ScienceDirect

Journal of Neuroscience Methods

journa l homepage: www.e lsev ier .com/ locate / jneumeth

omparison of extraction methods for peptidomics analysis of mouse brain tissue

nnemie Van Dijcka, Eisuke Hayakawab, Bart Landuytc, Geert Baggermanb, Debby Van Dama,alter Luytenc,d, Liliane Schoofsc, Peter Paul De Deyna,e,∗

Laboratory of Neurochemistry & Behaviour, Institute Born-Bunge, University of Antwerp, Department of Biomedical Sciences, Universiteitsplein 1, B-2610 Wilrijk, BelgiumProMeta, K.U.Leuven, Herestraat 49, B-3000 Leuven, BelgiumFunctional Genomics, and Proteomics, K.U.Leuven, Naamsestraat 59, B-3000 Leuven, BelgiumBiomedical Sciences Group, K.U.Leuven Medical School, Herestraat 49, B-3000 Leuven, BelgiumDepartment of Neurology/Memory Clinic, Middelheim General Hospital, ZNA, Lindendreef 1, B-2020 Antwerp, Belgium

r t i c l e i n f o

rticle history:eceived 8 February 2011ccepted 23 February 2011

eywords:europeptideass spectrometry

57Bl/6J

a b s t r a c t

The peptidome encompasses all the peptides present in a particular cell, tissue or organism at a par-ticular point in time. Neuropeptidomics studies the peptidome of the nervous system and will becomeincreasingly important in neuroscience research. Novel peptides can be discovered and, when applied todisease models, key players in pathophysiological mechanisms will be identified. That way, they can serveas drug targets or biomarkers. Presently, different extraction protocols are in use, but no consensus hasbeen reached on what fixation and extraction protocol is best suited for brain tissue. Therefore, in this arti-cle we compare different methods for quenching of proteolytic activity (snap-freezing of whole mouse in

ample preparationeptide extraction

liquid nitrogen immediately after cervical dislocation, freezing of the dissected brain in 2-methyl-butaneand heat denaturation of the tissue by microwave treatment) in combination with different extractionmethods. The protocol that combines submersion in liquid nitrogen with extraction in 0.25% acetic acidresults in the highest number of unique identifications, a high conservation of posttranslational mod-ifications, the best reproducibility between duplicate samples and the best comparison with formerstudies on mouse brain peptides. For these reasons, we recommend the use of this protocol in future

s.

neuropeptidomics studie

. Introduction

Neuropeptides are found throughout the entire nervous sys-em, where they can act as neurotransmitter or hormone. Theynfluence a wide range of physiological processes, including fear,ggression, circadian rhythms, learning and memory (Kastin,006). Consequently, levels of peptides in the brain reflect cer-ain information about physiological status (Svensson et al., 2007).adioimmunoassays and enzyme-linked immunosorbent assaysre commonly used to measure levels of neuropeptides. Although

hese approaches are sensitive, most antisera are not specific forsingle peptide and cross-react with other peptides that may dis-lay sequence similarities. Also, most antisera cannot distinguishetween posttranslational modifications (PTMs). In addition, these

Abbreviations: PTM, posttranslational modification; MB, 2-methyl-butane; LN,iquid nitrogen; MW, microwave; TFA, trifluor acetic acid; LC, liquid chromatogra-hy; MS, mass spectrometry.∗ Corresponding author at: Laboratory of Neurochemistry & Behaviour, Instituteorn-Bunge, University of Antwerp, Department of Biomedical Sciences, Univer-iteitsplein 1, B-2610 Wilrijk, Belgium. Tel.: +32 3 265 26 20;ax: +32 3 265 26 18.

E-mail addresses: peter.dedeyn@ua.ac.be, dedeyn@skynet.be (P.P. De Deyn).

165-0270/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.jneumeth.2011.02.023

© 2011 Elsevier B.V. All rights reserved.

techniques can only cope with a limited number of neuropeptidesat the same time and they are restricted to known peptides. Neu-ropeptidomics is the technological approach for detailed analysesof endogenous peptides from the brain, using mass spectrome-try and bioinformatics (Clynen et al., 2003; Boonen et al., 2008;Menschaert et al., 2010). It aims at the identification of as manypeptides as possible in one specific sample.

In the early days of peptidomics, the focus was on invertebrates,such as locusts and fruit flies (Clynen et al., 2001; Baggerman et al.,2002), but nowadays the focus is shifting to mammals, which aremore suitable as models for human diseases. For this purpose, espe-cially mice are interesting because of the large number of transgenicmodels established in this species. Additional knowledge about thefunction of peptides may help to understand the pathophysiologybehind different neurological diseases and form the basis for newtreatment strategies.

Most bioactive peptides are typically present at very low lev-els in comparison to major proteins, and so the breakdown of even

a miniscule fraction of one of these abundant proteins can over-whelm the weaker signals from the endogenous neuropeptidespresent in the extract (Skold et al., 2002; Svensson et al., 2003; Cheet al., 2005a). Therefore, different solutions have been proposed tokeep the relative concentration of neuropeptides compared to pro-

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32 A. Van Dijck et al. / Journal of Neu

ein degradation fragments as high as possible. One solution is tohoose tissues that are relatively high in peptide content and lown degrading enzymes, such as the pituitary (Desiderio, 1999), buthe situation is less favourable in other brain regions which peptideontent is highly relevant as well. In the past, several peptidomicstudies have been performed on the knockout Cpefat/fat strain thatacks carboxypeptidase E activity (Che et al., 2005a). In this model,ffinity columns can be used to preferentially enrich peptide-rocessing intermediates containing C-terminal basic residues.ajor drawbacks, however, are that this solution can only be used

or this mouse model, and that the results contain only a specificubset of peptides.

The most promising approach for preserving peptides and pre-enting the degradation of proteins is to keep the time betweeneath and the quenching of proteolytic enzyme activity as shorts possible. No consensus has been reached today regarding theest manner to achieve this. Different methods will be compared

n the present study: (1) immersion-fixation of the whole mousen liquid nitrogen immediately after cervical dislocation, (2) prele-ation of the brain followed by freezing it in 2-methyl-butane and3) conventional microwave irradiation (Che et al., 2005a, 2005b).

Extraction protocols should be kept as simple as possible, inrder to simultaneously extract maximal numbers of peptidesith different properties. Posttranslational processing, such as

midation or acetylation, can serve to regulate the resulting bioac-ive properties of a peptide (Perkins, 2006; Gualillo et al., 2006).herefore, the ideal extraction protocol should preserve these post-ranslational modifications. However, the most optimal extraction

ethod remains to be determined. For that reason, different pre-iously described extraction methods are compared in this studynd both their advantages and disadvantages are discussed.

. Materials and methods

.1. Euthanasia and brain dissections

C57Bl6/J male mice, aged 4 months (Charles River, Brussels,elgium) were sacrificed as needed for each peptide extraction pro-ocol. They were either killed by cervical dislocation or decapitationo keep time between death and quenching of proteolytic enzymectivity as short as possible. After cervical dislocation, brains wereemoved from the skull and immersed immediately in 2-methyl-utane (MB) cooled on dry ice to −40 ◦C, or the entire mouse wasnap-frozen in liquid nitrogen (LN). After the decapitation, thentire head was placed in the middle of a conventional microwaveven during 8 s at 750 W, where upon the brain was dissected out ofhe skull (MW). It was shown that conventional microwave treat-

ent is a valuable alternative to focused microwave irradiation, butuch more affordable and widely available (Che et al., 2005a). Also

fter freezing of the entire mouse in liquid nitrogen, the brain wasemoved. All experiments were carried out in compliance with theuropean Communities Council Directive (86/609/EEC) and proto-ols were approved by the Local Animal Ethics Committee.

The frozen brains were placed on a metal plate, which wasooled by a mixture of dry and wet ice; the microwave-treatedrains were not kept on ice and were dissected at ambient tem-erature. First, the frontal cortex was taken, followed by thetriatum; next the hypothalamus was removed. Finally, the hip-ocampus was dissected out. All tissue samples were stored inubes (Eppendorf, Hamburg, Germany) at −80 ◦C until extraction.

er combination of euthanasia method and peptide extraction pro-ocol, two mice were sacrificed, resulting in a total of twelve micesee under Section 2.2). For each mouse, the aforementioned fourrain regions were dissected and tissue samples were prepared andnalyzed separately, as biological replicates.

ce Methods 197 (2011) 231–237

2.2. Peptide extraction

Unless stated otherwise, all reagents were at least of analyti-cal grade and purchased from Merck, Darmstadt, Germany. Waterwas purified with a MilliQ system (Millipore, Brussels, Belgium).Protocol 1 was combined with all euthanasia methods because itseemed the most promising. All other protocols were combinedwith the euthanasia method found in the literature (or as usuallypracticed for the in-house protocol).

2.2.1. Protocol 1This extraction protocol was applied to the LN, MB and MW

brains. The protocol was based on the one of Nilsson and colleaguesand the similar protocol of Skold et al. (Skold et al., 2002; Nilssonet al., 2009). Tissue was homogenized by sonication in 0.25% aceticacid at 95 ◦C in a volume of 7.5 �l/mg of tissue with a Rapidis soni-cator (Ultrasonics, Shipley, UK) and incubated for 1 min at 95 ◦C ina hot water bath. Cell debris was removed by centrifugation dur-ing 40 min at 14,000 × g at 4 ◦C. The supernatant was transferredto Microcon YM-10 filtering devices (Millipore, Brussels, Belgium)and centrifuged at 14,000 × g during 60 min at 4 ◦C. The filtrate wastransferred to low-adhesion vials, dried in a SpeedVac (ThermoFisher, Breda, the Netherlands) and stored at −80 ◦C until use.

2.2.2. Protocol 2Tissues from the microwaved brains were processed accord-

ing to the second extraction protocol, modified from Zhang et al.(2008), whereby the steps necessary for the labeling method ofthat article, were omitted. In brief, brain tissue was sonicated twiceduring 20 s in 600 �l of ice-cold water. Next, the homogenates wereincubated at 70 ◦C in a water bath during 20 min. The samples werethen cooled on ice and acidified with 0.3% HCl to a final concentra-tion of 0.03% HCl. Afterwards, the homogenates were centrifuged at13,000 × g during 40 min at 4 ◦C. The supernatant was transferredto low-retention tubes (Bioplastics, Landgraaf, the Netherlands)and concentrated to 700 �l in a vacuum centrifuge and filtratedthrough Microcon YM-10 centrifugal filter units. Subsequently, thesamples were desalted using Sep-Pak Vac C18 cartridges (Waters,Zellik, Belgium) and elution solvent (13.4 M acetonitrile/0.01 M TFAin water). The cartridges were washed with 10% of this solvent inwater and the peptides were eluted off the column with 1 ml of thissolvent. The eluate was transferred to low-adhesion vials, dried ina SpeedVac and stored at −80 ◦C until use.

2.2.3. Protocol 3This protocol (Bora et al., 2008) was used for the LN-euthanized

mice. After preheating at 90 ◦C, 300 �l of milliQ water was addedto the frozen brain regions. Brain tissue was homogenized withthe aforementioned sonicator. Next, the tubes were placed intoa boiling water bath during 10 min. Afterwards; they were cen-trifuged at 14,000 × g during 5 min at 4 ◦C. The supernatant wastransferred to a new vial. Next, 300 �l of acidified acetone (40:6:1acetone/water/HCl) was added to the pellet and incubated for 1 hat 4 ◦C. Consecutively, this vial was vortexed during 1 min and cen-trifuged at 14,000 × g during 5 min. The supernatant was againcollected. Subsequently, 300 �l of 0.25% acetic acid was added tothe pellet and the sample was incubated during 1 h at 4 ◦C. Next,the tube was vortexed during 1 min and centrifuged at 14,000 × g

during 5 min. The supernatants of the prior 3 steps were pooled andfiltered through a Microcon YM-10 filter by centrifugation during1 h at 14,000 × g at 4 ◦C. The filtrate was subsequently transferred tolow-adhesion vials, dried in a SpeedVac and stored at −80 ◦C untiluse.

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.2.4. Protocol 4The MB brains were subjected to an extraction protocol that was

eveloped in-house. An extraction solution was prepared, consist-ng of 90% methanol, 1% formic acid and 9% milliQ water. Next,00 �l of this solution was added to each sample. The tissue wasomogenized with the Rapidis sonicator. The sample was cen-rifuged at 14,000 × g during 10 min at 4 ◦C. The supernatants wereransferred to low-adhesion vials, dried in a SpeedVac and storedt −80 ◦C until use.

.3. Mass spectrometry analysis

Capillary liquid chromatography (LC)/tandem MS experimentsere conducted using an Ultimate 3000 system run in nano-LC set-p (Dionex, Sunnyvale, USA) coupled to a Q-TOF hybrid quadrupoleime-of-flight MS (Micro-tofQ, Bruker, Bremen, Germany). Theample was dissolved in 10 �l of 5% acetonitrile in water andltered through a spin-down filter (Millipore). A 5 �l aliquot ofhis sample was injected onto a reversed-phase C18 precolumn�-guard column MGU-30 C18, LC Packings, Dionex). Sample load-ng was done at a flow-rate of 30 �l/min with water containing% acetonitrile and 0.1% formic acid. Loading took 10 min. Theeversed-phase loading-column was rinsed for another 5 min, thenhe reversed-phase trapping column was switched in-line with theapillary column, a Pepmap C18, 3 �m 75 �m × 150 mm nano col-mn (Dionex). Separation was conducted using a linear gradientrom 95% solvent A, 5% solvent B to 5% A, 45% B in 40 min (solvent: water, acetonitrile, formic acid; 94.9, 5, 0.1 (v,v,v); solvent B:ater, acetonitrile, formic acid; 19.9, 80, 0.1 (v,v,v)). The flow rateas set at 200 nl/min.

The LC system was connected in series with the electrospraynterface of the Q-TOF device. The column eluate was directedhrough a stainless-steel emitter (Proxeon, Odense, Denmark). Theeedle voltage was set at 1250 V. Nitrogen was used as nebuliz-

ng gas. Parent ions with charges of sufficient abundance wereutomatically selected for fragmentation as they eluted from theolumn by the software (Compass 1.3, Bruker) based on signalntensity and charge state (preferably double-charged). Argon wassed as the collision gas. The detection window in the survey scanas set from m/z 400 to 1500. Fragmentation spectra were acquired

rom m/z 50 to 2000.All MS and MS/MS spectra were automatically processed

background subtraction, smoothing and peak picking) using thelexAnalysis software (Bruker Daltonics) to generate peak list filesn MGF format. MS/MS Peak list files were submitted to a “Mas-ot” search (Matrix Science, London, UK) against the SwissProt1.6 database and taxonomy was set to Mus musculus (12,420equences), enzyme was set to none, which allows for the precur-or protein being cut at any position. Specific considerations haveeen taken into account to interpret Mascot scores correctly foreptidomics research (Menschaert et al., 2010).

. Results

.1. Tissue state after quenching

Microwaved brains were shrunk and often deformed. The leasteformation was obtained when heads were placed upside down

n the microwave oven. All samples reported in this article werehus treated in this way. Average sample weight is smaller for

icrowave-treated brain regions because of the evaporation ofater. However, brain regions could easily be separated, which

acilitated dissection. Removal of the brains from the mice sub-erged in liquid nitrogen was not easy. Skin and muscle had to be

craped off; the skull had to be removed piece by piece in order not

ce Methods 197 (2011) 231–237 233

to damage the brain, yet quickly enough in order for the brain notto thaw. Once the brain was taken out, the situation for dissectionwas comparable to that of the brains frozen in 2-methyl-butane.The solidity of the brain in frozen state is an advantage for dis-section, compared to fresh material. On the negative side, colordifferences between brain regions are harder to discern, which ren-dered identification of the different brain regions more difficult, butnot impossible.

3.2. Identification of peptides

Microwave treatment was combined with protocol 1 (MW1)and protocol 2 (MW2), submersion of the whole mouse in liq-uid nitrogen immediately after cervical dislocation combined withprotocol 1 (LN1) and protocol 3 (LN3), the brains frozen in 2-methylbutane immediately after dissection were extracted withprotocol 1 (MB1) and protocol 4 (MB4).

For comparison among samples and with studies in the lit-erature as well as for counting the number of unique identifiedpeptides, we considered peptides truncated by one amino-terminalor carboxy-terminal amino acid, not as additional peptide identifi-cations. This, because chances are high they arose by degradationduring the extraction process rather than being biologically distinctspecies. In addition, only sequences defined as peptides by SwissProt and derived from known peptide precursors were included fordata analysis. We want to stress that protein degradation fragmentsare thus not counted as peptides in this study.

In this study we identified up to 71 different peptides with onemethod. Among the identified peptides, 69% are novel peptides. Thenumber of identified peptides per extraction method was countedper brain region (Table 1). Each matching amino acid sequencewas counted only one time: either exactly the same amino acidsequence or one amino acid longer or shorter were considered asone count. The reproducibility between replicates can be found inthe last column. LN1 gave the highest number of identified peptidesadding all peptides found in at least one brain region, and MB1 thesecond highest number.

In this article, we evaluated how many of the detected peptidescould be found in the two identically treated brain regions. Thisgives us an indication on the reproducibility of the protocol. Thepercentage of reproducibility was the highest for LN1 with 95% onaverage over the different brain regions, and almost as high forMB1 with 92%; both are thus considerably higher than the othertested methods: 85% for MB4, 78% for MW1 and 49% for MW2.For LN3 this percentage could not be calculated since almost nopeptides could be identified. In the frontal cortex, 41 different pep-tides were found, 55 in hippocampus, 46 in hypothalamus and 27in striatum. Among those, 20 peptides could be identified in allfour brain regions. For differences between methods, see Table 1. Adetailed list of all peptides can be found in the Supplementary data,although some sequences had to be removed due to confidentialityreasons. However, all other relevant information for those peptidesis provided.

In our experiments, a number of peptide sequences correspondsto sequences previously observed in other brain peptidomics stud-ies: with a tolerance of one amino acid difference, or the sequencefound in our study corresponded partially to previously publishedpeptide sequence (Fig. 1). From the peptides found by Skold et al.(2002), 32% were also found with the LN1 protocol and the MB1protocol. Both these protocols are comparable to that of Skold et al.(2002). MB1 and LN1 reproduced on average 11% of peptides found

with protocols comparable to protocol 1 (Skold et al., 2002; Falthet al., 2006, 2007; Nilsson et al., 2009), while MW1 only reproduced9%. Protocol MW2 reproduced only 5% of peptides found in articlesthat used similar protocols (Che and Fricker, 2005; Che et al., 2005a,2005b; Zhang et al., 2008, 2010). Protocol LN3 could not reproduce

234 A. Van Dijck et al. / Journal of Neuroscience Methods 197 (2011) 231–237

Table 1Number of peptide identifications per sample preparation protocol.

Tissue fixation method Extraction protocol Brain region Number of identifications Percentage of reproducibility

2-Methyl-butane Protocol 1 Frontal cortex 12 100%Hippocampus 34 100%Hypothalamus 43 88%Striatum 33 82%

Protocol 4 Frontal cortex 6 100%Hippocampus 6 100%Hypothalamus 6 83%Striatum 7 57%

Microwave Protocol 1 Frontal cortex 4 100%Hippocampus 21 67%Hypothalamus 7 71%Striatum 13 77%

Procotol 2 Frontal cortex 9 11%Hippocampus 1 0%Hypothalamus 6 83%Striatum 3 100%

Snap frozen Protocol 1 Frontal cortex 27 89%Hippocampus 26 96%Hypothalamus 40 97%Striatum 39 97%

Protocol 3 Frontal cortex 0 NAHippocampus 1 100%Hypothalamus 0 NAStriatum 1 100%

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he number of peptide sequences found in at least one of the two duplicates is inast column, the percentage of peptide sequences found in both of the duplicate saonsidered the same. Percentage of reproducibility = (the number of peptides foudentified by that protocol in that brain region) × 100.

ny peptides found by Bora et al. (2008), despite the similarity of therotocols. This is to be expected, since different brain regions werenalyzed. The number of peptides derived from known bioactiverecursors is depicted in Fig. 2.

.3. Posttranslational modifications

We found the following PTMs in our samples: loss of glycinen combination with C-terminal amidation, acetylation of lysine,erine or cysteine, oxidation of methionine, N-terminal acetyla-

ig. 1. Correspondence with literature. In this graph, the correspondence of the peptidehe number of peptides that differ maximum 1 amino acid with earlier reported sequenorrespondence with literature. The black bars correspond to the total number of sequenervical dislocation, followed by freezing of the brain in 2-methyl-butane in combinationrain in 2-methyl-butane in combination with extraction protocol 4, MW1: decapitatiorotocol 1, MW2: decapitation followed by microwave treatment of the head in combinouse after cervical dislocation in combination with extraction protocol 1, LN3: submersith extraction protocol 3.

d, subdivided per euthanasia method, extraction protocol and brain region. In theis specified. Peptides differing by 1 amino acid amino- or carboxy-terminally are

both of brain regions treated by the same protocol/the total number of peptides

tion, and conversion of glutamine to N-terminal pyroglutamate.The number of PTMs found with each protocol is reported inTable 2. Specifications on the PTMs per peptide can be found inthe Supplementary table.

4. Discussion

The neuropeptidomics approach, as used in this study, has cer-tain advantages over the classical methods for studying peptides,such as enzyme-linked immunosorbent assay and radioimmunoas-

s found in our study is compared with earlier appearance in literature. In white,ces is shown. The grey columns represent the number of peptides with a partialces identified in our study with each protocol in the different brain regions. MB1:with extraction protocol 1, MB4: cervical dislocation, followed by freezing of the

n followed by microwave treatment of the head in combination with extractionation with extraction protocol 2, LN1: submersion in liquid nitrogen of the entireion in liquid nitrogen of the entire mouse after cervical dislocation in combination

A. Van Dijck et al. / Journal of Neuroscience Methods 197 (2011) 231–237 235

Fig. 2. Peptides derived from known bioactive precursors. The grey bars correspond to the number of peptides derived from known bioactive precursors. Those are precursors,which gave rise to known bioactive neuropeptides and/or start with a signal sequence that is responsible for secretion. The black bars represent the total number of peptidesin each brain region identified with the respective protocols. MB1: cervical dislocation, followed by freezing of the brain in 2-methyl-butane in combination with extractionp yl-bum W2: de cervicn on pro

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rotocol 1, MB4: cervical dislocation, followed by freezing of the brain in 2-methicrowave treatment of the head in combination with extraction protocol 1, M

xtraction protocol 2, LN1: submersion in liquid nitrogen of the entire mouse afteritrogen of the entire mouse after cervical dislocation in combination with extracti

ay or immunohistochemistry. In this study, we confirm that aarge number of peptides can be identified simultaneously, among

hich even novel peptides. The validity of the neuropeptidomicspproach is proven by the fact that known peptides, e.g. somato-tatin and corticotropin-like intermediary peptide, were detectedn addition to novel ones. Different variants of the same peptideone or several amino acids shorter) are easily discernible, whichs much harder with antibody-based techniques. PTMs are con-erved with several of our extraction methods and it is possibleo distinguish between the modified and unmodified peptide. Its very important to preserve PTMs in the light of their impor-ance for biological activity. Absence or presence of a specific PTMan mean the difference between an active or inactive variantf a peptide. C-terminal amidation and N-terminal acetylation,s found in our samples, often occur in vivo. They remove elec-

ric charge from peptide ends, thus allowing them to permeateetter through membranes. Those modifications also increase sta-ility, since they protect the peptide from aminopeptidases, as wells from synthetases. Amidation of peptides enhances activity ofeptide hormones (Kastin, 2006). Acetylation of side chains of in-

able 2osttranslational modifications.

Protocol Number of PTMs Percentage of total number of peptides

MB1 12 10%MB4 5 20%MW1 5 11%MW2 2 10%LN1 10 7%LN3 0 0%

he second column indicates the number of posttranslational modifications (PTMs)ound with each protocol. The percentage indicates which part of the total number ofeptides per protocol contained a posttranslational modification. MB1: cervical dis-

ocation, followed by freezing of the brain in 2-methyl-butane in combination withxtraction protocol 1, MB4: cervical dislocation, followed by freezing of the brain in-methyl-butane in combination with extraction protocol 4, MW1: decapitation fol-

owed by microwave treatment of the head in combination with extraction protocol, MW2: decapitation followed by microwave treatment of the head in combinationith extraction protocol 2, LN1: submersion in liquid nitrogen of the entire mouse

fter cervical dislocation in combination with extraction protocol 1, LN3: submer-ion in liquid nitrogen of the entire mouse after cervical dislocation in combinationith extraction protocol 3.

tane in combination with extraction protocol 4, MW1: decapitation followed byecapitation followed by microwave treatment of the head in combination with

al dislocation in combination with extraction protocol 1, LN3: submersion in liquidtocol 3.

between amino acids converts potentially cationic side chains intogroups, thus altering the charge distribution. In addition, acetyla-tion blocks ubiquitylation, and thus prolongs the half-life of themolecule (Walsh et al., 2005). Oxidation of methionine, on the con-trary, is more likely to be an artifact of the extraction procedure(Potgieter et al., 1997). In order to make assumptions on the preser-vation of PTMs, it would be necessary to study the PTMs presentbefore application of the extraction protocol. Inclusion of such aspecific study design was beyond the scope of the present article.

At present, there is no consensus on the sample preparationmethod of choice in neuropeptidomics. In this study we, there-fore, compared different proteolytic enzyme quenching methods incombination with several extraction methods. LN1 gave the high-est number of identifiable peptides with the best reproducibilitybetween duplicates. The second best protocol according to thesecriteria is MB1, with the advantage of easier dissection. The dif-ferent type of quenching method used in MW1 resulted in someadditional peptides. The same goes for MB4 with a completelydifferent extraction method. This makes MW1 and MB4 not the pri-mary choices as the most effective extraction methods, but thesemethods can be complementary if as much information as possi-ble about one brain region is to be gathered. LN3 and MW2 did notresult in much additional information. Moreover, protocols MB1and LN1 resulted in the highest conservation of PTMs. Correspon-dence between duplicates gives already an indication on the topic ofreproducibility, but in order to be fully conclusive, larger numbersof brains should be treated with the same protocol.

The different brain regions: frontal cortex, hypothalamus, hip-pocampus and striatum, were chosen because they are easy torecognize and to dissect, and thus could be sampled reproducibly. Inaddition, they play important roles in several neurological diseasesand are relatively rich in neuropeptide content. The LN1 methodextracts the highest number of peptides for all brain regions, exceptfor the hippocampus, where MB1 results in a slightly higher yield.Different peptides were identified in the specific brain regions,

although some peptides could be detected in several or all brainregions. From this we can conclude that our protocols are able toextract different types of peptides when present. It is noteworthythat not all peptides could be demonstrated in all brain regions.Given the fact that all of those brain regions have different biolog-

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cal functions, it seems logical that they do not contain the sameeuropeptides. But nevertheless, the question arises whether theifferences in our study reflect the natural occurring variation orot. Since the neuropeptidomics technology is quite new, not manytudies have been conducted on comparison of peptide presenceetween mouse brain regions. More classical antibody-based tech-iques are not able to discern between shorter or longer fragmentsf peptides. In addition, the protein expression of unknown pep-ides is of course not studied. Therefore, we chose to take a lookt the gene expression of the precursor at the mRNA level, sincehis information is the most broadly available for the peptides inur study. The comparison between the mRNA expression in theifferent brain regions and the identification in our study can beound in the Supplementary materials. Two major conclusions cane drawn: firstly, even when mRNA expression is low, the peptidean still be detected, secondly, the peptide is not always detectedven when mRNA expression level is high. Of course, this is notquantitative study, so we cannot discern whether the levels of

he identified peptides in our samples reflect the natural occur-ence. Quantitative studies should be performed to conclude onhis point. The fact that we seem to miss peptide identification evenhen expression level is high, is probably due to the selection of

he most abundant peaks for fragmentation in the mass spectrom-ter. When too many abundant peaks arise at the same moment,ome of them are not fragmented and thus not identified. Futureechnical improvements of mass spectrometers can bring solace tohis problem.

Some differences in reported peptides between this study andreviously published studies are probably due to slight differences

n sample treatment and extraction protocols. An important differ-nce between our experiment and that of the Fricker group, is thathey often used Cpefat/fat mice, which allows specific enrichmentf Lys- and Arg-extended intermediates, resulting in a high num-er of identifiable peptides (Che et al., 2005b; Zhang et al., 2008).nfortunately, this is only an advantage for this type of peptides,hile peptides not cleaved by carboxypeptidase E are completely

bsent. Our protocol gives thus a broader spectrum of peptides andan also be applied to normal mice or other transgenic models.rom the protocol of Zhang et al. (2008), the steps necessary forhe labeling were omitted, which could, unexpectedly, also haven impact on the outcome. We were not able to reproduce any ofhe peptides found by Bora et al. (2008), but this could be explainedy the fact that they dissected out a very specific brain region, theupraoptic nucleus, which is very rich in peptide content and con-ains a very specific set of peptides. The supraoptic nuclei of 12–17nimals were pooled, while in our experiment tissue of a singlenimal was used. Despite the differences in material and methods,he literature comparison is still useful to show that our method isble to identify partially the same peptides as previously publishedxperiments.

As it is important to inhibit proteases and peptidases as fast asossible, the time until inhibition in the very center of the brain

s important. When a mouse is submerged in liquid nitrogen, itakes 80 s for the temperature to reach −20 ◦C in the very center ofhe brain (7 mm from the skin surface) (Roos, 1999). Time betweenervical dislocation and submersion is only a few seconds. With theicrowave treatment, 80 ◦C in the brain is reached after 8 s (Che et

l., 2005a), and time between death and microwaving was around0 s. The microwave treatment is thus faster, but the numbers ofpecific peptides are substantially lower in samples heated in theicrowave oven, because some peptides may be labile under heat

reatment (Che et al., 2005a). Therefore, a specific set of peptides isissed when using the microwave treatment.Since abundant peptides mask the less abundant ones, degra-

ation fragments of proteins should be avoided. Peptides that areerived from known bioactive precursors are more likely to be

ce Methods 197 (2011) 231–237

naturally occurring peptides and not degradation fragments. Abioactive precursor was defined as a precursor which is known togive rise to bioactive neuropeptides. In addition, precursors thatstart with a signaling sequence that indicates that the protein issecreted have higher chances to be active outside of the cell. There-fore, those precursors are also counted as bioactive. Again, LN1 isthe best scoring protocol on this criterion, with MB1 as a goodsecond, as can be seen if Fig. 2.

Along the same lines, there is the problem of contaminationwith fragments of blood proteins. Gelman et al. (2010) showed thatpeptides derived from alpha- and beta-hemoglobin are present inmouse brain. This set of peptides is largely distinct from those inblood, indicating that these are endogenous signaling moleculesand not derived from blood that is present in the brain. Therefore,we considered hemoglobin also as a known precursor of bioac-tive peptides. Nevertheless, we are concerned here with naturallyoccurring peptides and proteolysis of hemoglobin before quench-ing of proteolytic enzyme activity should be avoided. In the caseof cervical dislocation and submersion in liquid nitrogen, the dam-age of the blood circulation only occurs after the quenching, whichprobably results in less degradation fragments of hemoglobin fromthe blood. In addition, sequence similarities were found betweenthe hemoglobin-derived peptides with the LN1 protocol and thebrain peptides described by Gelman et al. (2010). The hemoglobinfragments found with this method are thus likely to be endoge-nously present in brain tissue. In order to gain certainty about thishypothesis, additional experiments should be performed wherebyall blood is removed from the brain by ante mortem cardiac perfu-sion.

Independently from the method of euthanasia, protocol 1 resultsclearly in the highest number of identifiable peptides. This is a verysimple and short extraction method using 0.25% acetic acid in wateras extraction solvent. It is probably no coincidence that the leastcomplex extraction process is the best. Some peptides are presentin very low amounts, and in every sample preparation step, peptidemolecules are lost. This results in a final concentration below thedetection limit. Acetic acid and formic acid are weak organic acids,while peptides are zwitter ions. The addition of an acid renders thepeptides positively charged, which makes them (more) soluble inwater. The counter ions of strong mineral acids such as HCl (as usedin protocol 2 and 3) can form strong ion pairs with the positivelycharged analytes, decreasing sensitivity. Trifluor acetic acid (TFA)is an organic acid, however due to its very low pKa, it has the samedisadvantage as HCl. Due to the strong binding of Cl− and TFA tothe peptides, it is not easy to get rid of these acids prior to analysisin the MS. The C18 columns, used in protocol 2 are intended to getrid of salts and the HCl, but when the TFA or Cl− are bound to thepeptides, they will attach to the solid phase via the peptides, andthey will not be washed off. The binding of the acids to the peptidesalso prevents evaporation of the acids in the SpeedVac. Formic acid(protocol 4) is compatible with MS, but probably the peptides aretoo charged because of the addition of acid to dissolve in the 90%methanol solution. Nevertheless, this protocol gave good resultsfor the extraction of peptides from the central nervous system ofarthropods (Baggerman et al., 2002) and nematodes (Husson et al.,2010). Another possibility is that the methanol also extracts fat,which may interfere with the ionization of the peptides.

Since protocol LN1 resulted in the highest number of iden-tifications, the best reproducibility between duplicate samplesand of results found in other published studies, as well as ahigh conservation of PTMs, we recommend protocol LN1 for neu-

ropeptidomics studies in mice. The neuropeptidomics analysisof mouse brain holds many promises for the unraveling of thepathophysiology of a wide array of neurological diseases. Dif-ferential analysis between diseased and healthy brain, e.g. intransgenic mouse models, and correlation with the symptoms

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esearch.

cknowledgments

We want to thank Tom Vandekerckhove and Gerben Menschaertor help with the bioinformatics analysis. This work was supportedy the Fund for Scientific Research-Flanders (FWO grant G.0164.09nd FWO grant G0437.10), Interuniversity Poles of AttractionIAP Network P6/43), the Flemish Institute supporting Scientific-echnological Research in industry (IWT; SBO grant IWT-50164),ethusalem excellence grant of the Flemish Government, agree-ent between Institute Born-Bunge and University of Antwerp,

he Medical Research Foundation Antwerp, the Thomas Riellaertsesearch fund, and Neurosearch Antwerp. DVD is a postdoctoralellow of the FWO. BL acknowledges the industrial research foun-ation of K.U.Leuven.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.jneumeth.2011.02.023.

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