Peptide profiling of Internet-obtained Cerebrolysin using high performance liquid chromatography - electrospray ionization ion trap and ultra high performance liquid chromatography

Short communicationDrug Testing

and Analysis

Received: 19 February 2015 Revised: 28 April 2015 Accepted: 28 April 2015 Published online in Wiley Online Library

(www.drugtestinganalysis.com) DOI 10.1002/dta.1817

Peptide profiling of Internet-obtainedCerebrolysin using high performance liquidchromatography – electrospray ionization iontrap and ultra high performance liquidchromatography – ion mobility – quadrupoletime of flight mass spectrometryBert Gevaert,a Matthias D’Hondt,a Nathalie Bracke,a Han Yao,a

Evelien Wynendaele,a Johannes Petrus Cornelis Vissers,b Martin De Cecco,b

Jan Claereboudtb and Bart De Spiegeleera*

Cerebrolysin, a parenteral peptide preparation produced by controlled digestion of porcine brain proteins, is an approved noot-ropicmedicine in some countries. However, it is also easily and globally available on the Internet. Nevertheless, until now, its exact
chemical composition was unknown. Using high performance liquid chromatography (HPLC) coupled to ion trap and ultra highperformance liquid chromatography (UHPLC) coupled to quadrupole-ion mobility-time-of-flight mass spectrometry (Q-IM-TOFMS), combined with UniProt pig protein database search and PEAKS de novo sequencing, we identified 638 unique peptides inan Internet-obtained Cerebrolysin sample. The main components in this sample originate from tubulin alpha- and beta-chain,actin, and myelin basic protein. No fragments of known neurotrophic factors like glial cell-derived neurotrophic factor (GDNF),neurotrophin nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF) werefound, suggesting that the activities reported in the literature are likely the result of new, hitherto unknown cryptic peptides withnootropic properties. Copyright © 2015 John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.

Keywords: high definitionmass spectrometry; Cerebrolysin; cryptic peptides; ion trapmass spectrometry; ionmobility separation, Internet-obtained medicines

* Correspondence to: Bart De Spiegeleer, Drug Quality and Registration (DruQuaR)group, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg460, Ghent B-9000, Belgium. E-mail: [email protected]

a Drug Quality and Registration (DruQuaR) group, Faculty of PharmaceuticalSciences, Ghent University, Ottergemsesteenweg 460, Ghent, B-9000, Belgium

b Waters, ConneXion Business Park, Brusselsesteenweg 500, Zellik, B-1731, Belgium

Introduction

Due to globalization, nowadays it is easy to obtain medicines fromall over the world through Internet ordering. However, this gene-rally uncontrolled distribution not only has trade-economic conse-quences, but can also negatively affect the patient’s health. TheseInternet-obtainedmedicines are often illegal (substandard, falsified,not globally authorized) and have an increased risk of inappropriateuse by the patient as well as an insufficiently assured quality.[1,2]

They are not only a problem in Third World countries, but evenmore so in Western countries.[3,4] An example is Cerebrolysin, regis-tered in some Asian (e.g. Chinese registration number: H20100443)and eastern European countries (e.g. Lithuanian registration num-ber: LT/1/96/0820/001), but not approved in most countries world-wide. Nevertheless, it can easily be ordered over the Internetthroughout the world.

Cerebrolysin is a parenteral peptide preparation, produced bycontrolled digestion of lipid-free porcine brains. The supplier statesthat it is composed of 75% free amino acids and 25% lowmolecularweight proteins (<10 000 Da), based on the free nitrogen

Drug Test. Analysis (2015)

content.[5] Also relatively high levels of magnesium, potassium,phosphorus, and selenium are present.[6] However, a compositionof 80% peptides and only 20% amino acids has also beenreported.[7] Furthermore, inconsistency about the identity and ori-gin of the peptides present in the drug is an issue and results inquestions about the product’s quality. It is stated that Cerebrolysincontains peptides from digested porcine brain[8–10] or digested por-cine brain proteins,[7,11,12] but also neuropeptides,[13–16] fragmentsof known neurotrophic factors (like NGF) and peptides (enkephalin,orexin, and galanin)[17] or even unprocessed neurotrophic factors

Copyright © 2015 John Wiley & Sons, Ltd.

B. Gevaert et al.

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like BDNF[18] are mentioned to be the active components ofCerebrolysin.The product, first developed in Russia a few decades ago, is

claimed to have a positive effect on various central disorders like is-chemic stroke and vascular dementia.[7,19] In vitro experiments oncells,[8,9,11] as well as in vivo animal experiments[14,15,17,18,20–29] andhuman clinical trials,[12,13,16,30–36] report various effects, with a highnumber of results written in Russian and published in localjournals[10,17,22–24,30–39]; several other studies are sponsored by ordone in collaboration with the manufacturer.[8,14,26,28,29,40] Despitethis research, meta-analysis of the currently available vascular de-mentia and ischemic stroke clinical trials data indicate that thereis not enough evidence to recommend the medicine as a generalevidence-based standard therapy for these diseases.[7,19] Althougha few of the active peptides in Cerebrolysin were reported byGromova et al.,[17] these findings were not confirmed by other re-searchers; until now, no full chemical characterization of the pep-tide content has been reported. However, as the reported effectsof the medicine resemble those of some well-known neurotrophicfactors, like ciliary neurotrophic factor (CNTF), glial cell-derived neu-rotrophic factor (GDNF), neurotrophin nerve growth factor (NGF)and brain-derived neurotrophic factor (BDNF), it can be assumedthat peptide fragments of these proteins are the active substancesof the product.[40]

As the chemical composition of a product of this origin is very im-portant for consistent quality, safety and efficacy and seeing thevarious contradictory results reported about its content, this studyaims to clarify its peptide composition. Two different massspectrometry (MS) analyzer techniques were used and compared,i.e., the traditional ion trap (IT) and the novel high definitionquadrupole-time-of-flight which encompasses an ion mobility sep-aration (HDMS Q-IM-TOF).

Materials and methods

Materials

Cerebrolysin (5 x 5 mL, Ever NeuroPharma, lot: 12018605) was ob-tained through an Internet order. High performance liquid chroma-tography (HPLC) gradient grade acetonitrile was purchased fromFisher scientific (Aalst, Belgium). Liquid chromatography-massspectrometry (LC-MS) grade formic acid was purchased from Fluka(Buchs, Switzerland). Water was purified using an Arium 611 purifi-cation system (Sartorius, Gottingen, Germany) yielding ≥ 18.2MΩ×cm quality water.

Methods

HPLC-ESI-ion trap MS

The liquid chromatography-ultraviolet/mass spectrometry (LC-UV/MS)apparatus consisted of a Spectra System SN4000 interface, aSpectra System SCM1000 degasser, a Spectra System P1000XRpump, a Spectra System AS3000 autosampler and a Finnigan LCQClassic ion trap mass spectrometer in positive ion mode (allThermo, San José, CA, USA) equipped with a Waters 2487UV–detector (all Waters, Milford, MA, USA) and Xcalibur 2.0 soft-ware (Thermo, San José, CA, USA). Electrospray ionization (ESI)was conducted using a needle voltage of 4.5 kV. Nitrogen was usedas the sheath and auxiliary gas with the heated capillary set at280°C. CID experiments employed helium with a collision energyset at 35%. Twenty μL of the undiluted sample was injected on a

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Vydac organic acid column (4.6 x 250 mm, 5 μm; Grace Vydac,Hesperia, CA, USA) at room temperature with a mobile phaseconsisting of 0.1% w/v formic acid in water (MPA) and 0.1% w/vformic acid in acetonitrile (MPB). After a 5-min isocratic period with100% MPA, a linear gradient was started from 100% MPA to 80%MPA in 25 min. The flow rate was set at 1.0 mL/min. The data wasautomatically processed using the Xcalibur program componentBioWorks 3.2 (all Thermo, San José, CA, USA) and the peptide se-quences were compared with the UniProt Sus scrofa (porcine) pro-teins according to the mass spectrum. Conditions of peptideselection included a fragment ion tolerance of 1.0 amu andSEQUEST Xcorr versus charge state parameters set at 1.5 and 2.1for single- and double-charged peptides, respectively. Finally, themass spectra of all retained peptides were manually checked. Thegene distribution of the identified proteins was controlled usingthe BioGPS platform.[13] The obtained sequences were also com-pared with the sequences of the neurotrophic factors BDNF, GDNF,NGF, and CNTF.

UHPLC-QTOF HDMS analysis

The LC-MS equipment consisted of an Acquity I-class UPLC coupledto a SYNAPT G2-Si (all Waters Corporation, Milford, MA, USA). TheAcquity HSS C18 column (2.1 x 150 mm, 1.8 μm; Waters, Milford,MA, USA) was placed in a column oven and the temperature wasset at 40°C; 0.5 μL of the undiluted Cerebrolysin sample wasinjected. MPA consisted of 0.1% w/v formic acid in water andMPB of 0.1% w/v formic acid in acetonitrile. After a 3-min isocraticperiod with 100% MPA, a linear gradient was started from 100%MPA to 60%MPA in 27min with a flow rate of 250 μL/min. The datawere acquired using four different acquisition modes: (1) DIA dataindependent analysis (MSE) in ESI+ with a mass range from 50–2000 m/z, a scan time of 0.3 s and collision energy ramp (trap) of4 eV (low energy) and 15–40 eV (high energy); (2) IM-DIA ionmobil-ity assisted data independent analysis (HDMSE) in ESI+ with a massrange from 50 to 2000m/z, a scan time of 0.3 s and collision energyramp (trap) of 4 eV (low energy) and 20–50 eV (high energy); (3)DDA data-dependent analysis in ESI+ with a mass range from 50to 2000 m/z, a survey scan time of 0.2 s, a MS/MS scan time of 0.1s, peak detection with charge state 2+ to 5+ and collision energyramp (trap) of 5–10 eV (low mass) and 60–70 eV (high mass); and(4) enhanced (product ion intensity) DDA in ESI+ with a mass rangefrom 50 to 5000m/z, a survey scan time of 0.2 s, a MS/MS scan timeof 0.1 s, peak detectionwith charge state 2+ to 5+ and collision ener-gy ramp (trap) of 6–9 eV (low mass) and 147–183 eV (high mass).The data were processed using the ProteinLynx GlobalSERVER3.0.1 (PLGS; Waters Corporation, Milford, MA, USA; DIA andIM-DIA) and Mascot (DDA and enhanced DDA) using the UniProtSus scrofa, reviewed proteins. Also a UniProtMammalia and Bacteriaprotein databases search was performed to evaluate the presenceof specific non-pig related peptides. Data were further visualizedand manually analyzed using Scaffold 4 (Proteome Software Inc.,Portland, OR, USA) with a peptide threshold of 95% and a proteinthreshold of 20% resulting in a false discovery rate (FDR) of < 1%for the Sus scrofa search and 2.39% for the Mammalia and Bacteriasearch. The peptide threshold refers to the Scaffold peptideprobability rate, which is an algorithm based on PeptideProphet,that automatically validates peptide assignments to tandemmass spectrometry (MS/MS) spectra made by database searchprograms.[41] Further, the Scaffold reported MS/MS total ion current(TIC) was used for semi-quantification. Next to the database search,de novo sequencing was done for the identification of peptides

15 John Wiley & Sons, Ltd. Drug Test. Analysis (2015)

Peptide profiling of cerebrolysin using HPLC-IT MS/MS and UHPLC-Q-IM-TOF

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using PEAKS (version 7.0, Bioinformatics Solutions Inc., Waterloo,ON, Canada) with the following parameters: parent ion mass toler-ance = 10.0 ppm; fragment ion mass tolerance = 0.1 Da; enzyme =nonspecific; maximum allowed variable post-translational modifica-tion (PTM) per peptide = 1; ALC-score> 60%. Finally, BLAST searchesof de novo identified peptide sequences using pig proteinsequences were performed and the identifications evaluated basedon the number of alignments expected by chance (E-value) and thehighest alignment score (Max-score).

Results

As described in the Methods section, the sample was directly ana-lyzed by LC-MS without a previous purification or enzymatic diges-tion. The TIC chromatograms are presented in Figure 1. The

Figure 1. Ion trap MS and enhanced DDA Q-IM-TOF MS total ion chromatogra

Figure 2. Analytical process flow chart.

Drug Test. Analysis (2015) Copyright © 2015 John Wiley

analytical steps and data-evaluation are visualized in a process flowdiagram (Figure 2).

HPLC-ion trap MS

After applying the Δm and Xcorr filters and manual inspection ofthe MS spectra, 13 peptides were retained (Table 1). The peptidelength ranges from 6 to 15 amino acids, with an average lengthof 9.2 amino acids; the peptide mass ranges from 682.34 Da to1889.05 Da, with an average mass of 1015.89 Da. Using UniProt’spig brain proteins, the peptides could be assigned to seven differ-ent proteins, with myelin basic protein (MPB) and beta-actin (ACTB)being the most important ones with respectively five and threepeptides. Also peptides originating from tubulin alpha 1-A (TBA1A),tubulin alpha 1-B (TBA1B), phosphatidylinositol-4,5-bisphosphate3-kinase catalytic subunit gamma (PK3CG) and the uncharacterized

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Table 1. Ion trap MS identified peptides

Peptide UniProt Reference MH+ ΔM z XCorr

VGEGMEEGE TBA1A_PIG Tubulin alpha-1A chain 936.3615 �0.0245 1 1.7021

GAQDAQGT MBP_PIG Myelin basic protein 747.3268 �0.0033 1 1.5443

YQPPTVVPGGDLA TBA1B_PIG Tubulin alpha-1B chain 1313.674 �0.4267 1 1.8655

YQRVRVENVCKRLNL I3LNP3_PIG Uncharacterized protein (Fragment) 1890.049 0.0317 3 2.0945

DFEQEM ACTB_PIG Actin, cytoplasmic 798.2974 �0.0307 1 1.5430

VAPEEHPV ACTB_PIG Actin, cytoplasmic 877.4414 �0.0128 1 1.7193

TEAPLNPK ACTB_PIG Actin, cytoplasmic 869.4727 �0.0756 1 1.8144

LLDAVPL F1S3X7_PIG Uncharacterized protein 740.4553 �0.0277 1 1.6059

TPPPSQG MBP_PIG Myelin basic protein 683.3359 �0.1331 1 1.7287

GAEGQKPGFG MBP_PIG Myelin basic protein 947.4581 �0.0268 1 2.2889

KDEVLSH PK3CG_PIG Phosphatidylinositol 4,5-bisphosphate

3-kinase catalytic subunit gamma

827.4258 �0.6178 2 2.1353

FSWGAEGQKPGFG MBP_PIG Myelin basic protein 1367.638 �0.6108 2 3.3411

SWGAEGQKPGFG MBP_PIG Myelin basic protein 1220.569 �0.4711 2 3.3486

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proteins I3LNP3 and F1S3X7, all originating from the pig, werefound. As the spectra were compared with UniProt’s pig brain pro-teins, no peptides originating from other than pig proteins wereidentified. The distribution of the identified proteins in the brain,verified by BioGPS, suggests that Cerebrolysin indeed contains pep-tides derived from pig brain proteins, as claimed by themanufacturer.[42] The selective database search for the known neu-rotrophic factors BDNF, GDNF, NGF and CNTF did not result in anyhits using the applied Δm and Xcorr filters.

UHPLC-Q-IM-TOF

The analysis of the sample on the high resolution mass spectrome-ter, which creates the possibilities of analyzing samples not onlybased on mass and charge but also, due to the extra ion mobilityseparation, differentiate in size and shape, resulted in a morediverse result. An example enhanced DDA chromatogram ispresented in Figure 1. The spectra were compared with the PLGSdatabase search (DIA and IM-DIA) and Mascot search (DDA andenhanced DDA) algorithm using pig proteins. By DDA, 36 peptides,including 9 unique sequences which were only identified using thisacquisition mode, from 18 different proteins were found, whereas38 peptides, including 10 unique sequences, were found from 20distinct proteins when using the extra ion mobility separation facil-ity. Even more peptides were found with the DIA acquisition mode:15 peptides, from which one was only identified with this acquisi-tion mode, originating from 7 different proteins, could be identi-fied. This number was increased to 89 peptides, including 56unique sequences, from 14 proteins when using the extra ionmobility separation. Overall, based on the database search, 119unique peptide sequences were found, originating from 34 differ-ent proteins. The average peptide masses, obtained using the dif-ferent techniques, were very similar, i.e., 874.32 Da for DDA,878.20 Da for enhanced DDA, 994.69 Da for DIA and 895.92 Dafor IM-DIA. Moreover, the de novo sequencing of the enhancedDDA data resulted in the identification of 517 peptides from 491proteins. A list with all 633 peptide sequences, tentatively identifiedwith LC-Q-IM-TOF MS, is given in the supplementary information.The results confirmed the HPLC-ion trap MS data: the highest num-ber of peptides originates from tubulin alpha- and beta-chain, actinand myelin basic protein. The sequences of these four proteins,with the identified peptide sequences highlighted, are presentedin Table 2. For each acquisition method, the 10 most abundant


peptides, including their normalized occurrence, are presented inTable 3. They represent 51.9%, 88.5%, 45.4%, and 57.9% of theMS/MS total ion current using IM-DIA, DIA, enhanced DDA, andDDA, respectively. In all acquisition methods, MBP 115–126(SWGAEGQKPGFG) was with 10.5% (IM-DIA), 17.7% (DIA), 9.9%(enhanced DDA), and 15.8% (DDA) of the MS/MS total ion current,the most abundant peptide in the Cerebrolysin sample. The corre-sponding enhanced DDA Q-IM-TOF and ion trap MS/MS spectraare presented in Figure 3. Also other EGQKPGF containing se-quences were highly abundant, representing 29.6% (IM-DIA),33.7% (DIA), 14.9% (enhanced DDA), and 33.1% (DDA) of the totalMS/MS ion current. Although identical sequences were identifiedwith each mass analyzer and acquisition method, each techniqueshowed clearly its value as demonstrated by the number of uniquesequences identified (Figure 2). These numbers clearly demonstratethe added-value of ion mobility separation, especially in combina-tion with data-independent analysis (HD-MSE) for the analysis ofthese complex samples.

By comparing the spectra with the Mammalia database, all iden-tified sequences could be linked to pig derived proteins. As a result,there is no demonstrated evidence that tissues from otherMammalia, for example bovine brains, were used for the prepara-tion of this pharmaceutical product. The spectra were also com-pared with the UniProt Bacteria proteins to control for thepresence of bacterial contaminations which would indicate a failurein Good Manufacturing Practices (GMP). In total, only two spectracould be linked to a bacterial peptide (AVAMELDIP andITAGAAGPL). However blast search or de novo sequencingindicated that they could originate from pig proteins as well.

Discussion

Cerebrolysin is a complexmixture, consisting of mainly amino acidsand peptides. In this study our goal was to characterize the peptidecontent using mass spectrometry, the state-of-the-art technique inpeptidomics. Different MS techniques and acquisition modes wereused in the analysis of this complex Internet peptide sample. First, adifferentiation can be made based on the mass analyzer. Next tothe well-known low-resolution ion trap, also a high resolution QTOFmass analyzer was used. With this HR-mass spectrometer, twomainacquisition methods were applied: DDA and DIA. DDA is a tradi-tional MS/MS approach where a parent mass is selected based on


Table 3. List of the 10 most abundant peptides identified with each QTOF acquisition mode with their abundance

IM-DIA DIA

UniProt access number Sequence %a UniProt access number Sequence %a

F1RLM1_PIG PGPKGSVGGMGLPGT 2.3% ACTS_PIG AVFPSIVGRPR 13.0%

F1SEK0_PIG LEERTDEGDPV 3.3% ACTS_PIG VFPSIVGRPR 7.8%

F1SRL9_PIG SGPAAGGEAPK 3.7% F1RLM1_PIG PGPKGSVGGMGLPGT 6.3%

I3LVD5_PIG VAPEEHPV 8.6% I3LVD5_PIG VFPSIVGRP 7.8%

MBP_PIG FSWGAEGQKPGFG 4.1% MBP_PIG FSWGAEGQKPGFG 10.3%

MBP_PIG GAEGQKPGFG 10.3% MBP_PIG SWGAEGQKPG 5.7%

MBP_PIG SWGAEGQKPGFG 10.5% MBP_PIG SWGAEGQKPGFG 17.7%

TBA1A_PIG VGEGMEEGE 3.9% TBA1A_PIG NYEPPTVVPGGD 8.5%

TBA1A_PIG VGEGMEEGEF 2.2% TBA1A_PIG NYEPPTVVPGGDLA 6.6%

TBB5_PIG SLGGGTGSGMGT 3.1% TBB5_PIG VDLEPGTMDSV 4.8%

Enhanced DDA DDA

UniProt access number Sequence %a UniProt access number Sequence %a

ACTS_PIG AVFPSIVGRP 2.6% ACTS_PIG AVFPSIVGRPR 4.6%

ACTS_PIG TEAPLNPK 9.0% ACTS_PIG TEAPLNPK 8.9%

F1S263_PIG ELKSLEEEL 3.0% B7TY10_PIG QGPPLQQRPTQ 2.6%

I3LBD7_PIG LDKPIQEV 3.7% F1SRL9_PIG SGPAAGGEAPK 6.6%

I3LJE2_PIG DHVVPEPGTS 3.7% I3LMX2_PIG QHGRPQDENPV 3.7%

K7GLT8_PIG KIPVGPETLG 2.6% K7GLT8_PIG KIPVGPETLG 2.3%

MBP_PIG FSWGAEGQKPG 3.1% MBP_PIG FSWGAEGQKPGFG 3.9%

MBP_PIG SWGAEGQKPGFG 9.9% MBP_PIG GAEGQKPGFG 6.1%

TBB5_PIG HSLGGGTGSGMGT 3.7% MBP_PIG SWGAEGQKPGFG 15.8%

TBB5_PIG LVDLEPGTMDSV 4.2% MBP_PIG WGAEGQKPGFG 3.4%

anormalized MS/MS total ion current

Table 2. Protein sequences with identified peptides highlighted

Protein name Sequence

TBA1A_PIG Tubulin

alpha chain

MRECISIHVGQAGVQIGNACWELYCLEHGIQPDGQMPSDKTIGGGDDSFNTFFSETGAGKHVPRAVFVDLEPTVIDEVRTGT

YRQLFHPEQLITGKEDAANNYARGHYTIGKEIIDLVLDRIRKLADQCTGLQGFSVFHSFGGGTGSGFTSLLMERLSVDYGKK

SKLEFSIYPAPQVSTAVVEPYNSILTTHTTLEHSDCAFMVDNEAIYDICRRNLDIERPTYTNLNRLIGQIVSSITASLRFDGALN

VDLTEFQTNLVPYPRAHFPLATYAPVISAEKAYHEQLSVAEITNACFEPANQMVKCDPRHGKYMACCLLYRGDVVPKDVN

AAIATIKTKRTIQFVDWCPTGFKVGINYEPPTVVPGGDLAKVQRAVCMLSNTTAIAEAWARLDHKFDLMYAKRAFVHWYV

GEGMEEGEFSEAREDMAALEKDYEEVGVDSVEGEGEEEGEEY

TBB5_PIG Tubulin

beta chain

MREIVHIQAGQCGNQIGAKFWEVISDEHGIDPTGTYHGDSDLQLDRISVYYNEATGGKYVPRAILVDLEPGTMDSVRSGPFGQ

IFRPDNFVFGQSGAGNNWAKGHYTEGAELVDSVLDVVRKEAESCDCLQGFQLTHSLGGGTGSGMGTLLISKIREEYPDRIM

NTFSVVPSPKVSDTVVEPYNATLSVHQLVENTDETYCIDNEALYDICFRTLKLTTPTYGDLNHLVSATMSGVTTCLRFPGQLN

ADLRKLAVNMVPFPRLHFFMPGFAPLTSRGSQQYRALTVPELTQQVFDAKNMMAACDPRHGRYLTVAAVFRGRMSMKE

VDEQMLNVQNKNSSYFVEWIPNNVKTAVCDIPPRGLKMAVTFIGNSTAIQELFKRISEQFTAMFRRKAFLHWYTGEGMDEM

EFTEAESNMNDLVSEYQQYQDATAEEEEDFGEEAEEEA

I3LVD5_PIG Actin,

cytoplasmic

MEEEIAALVIDNGSGMCKAGFAGDDAPRAVFPSIVGRPRHQGVMVGMGQKDSYVGDEAQSKRGILTLKYPIEHGIVTNWDD

MEKIWHHTFYNELRVAPEEHPVLLTEAPLNPKANREKMTQIMFETFNTPAMYVAIQAVLSLYASGRTTGIVMDSGDGVTHT

VPIYEGYALPHAILRLDLAGRDLTDYLMKILTERGYSFTTTAEREIVRDIKEKLCYVALDFEQEMATAASSSSLEKSYELPDGQV

ITIGNERFRCPEALFQPSFLGMESCGIHETTFNSIMKCDVDIRKDLYANTVLSGGTTMYPGIADRMQKEITALAPSTMKIKIIAP

PERKYSVWIGGSILASLSTFQQMWISKQEYDESGPSIVHRKCF

MBP_PIG Myelin

basic protein

ASQKRPSQRHGSKYLASASTMDHARHGFLPRHRDTGIDSLGRFFGADRGAPKRGSGKDGHHAARTTHYGSLPQKAQHGRP

QDENPVVHFFKNIVTPRTPPPSQGKGRGLSLSRFSWGAEGQKPGFGYGGRAPDYKPAHKGLKGAQDAQGTLSKIFKLGGR

DSRSGSPMARR


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the programmed acquisition settings like repeat count, repeat du-ration, minimum MS signal and dynamic exclusion, after which itis fragmented.[43] DIA is a relatively new acquisition method, intro-duced by Bateman et al. in 2002,[44,45] using two scanning functions


at the same time, one with low and one with high collision energy,collecting precursor and product ion data simultaneously. For bothacquisition methods, runs with and without ionmobility separationwere conducted, creating an extra dimension where molecules


Figure 3. Ion trap and enhanced DDA Q-IM-TOF MS/MS spectra of SWGAEGQKPGFG.

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with the same m/z can be separated based on their cross sectionalarea which is a function of their conformation and size.[46]

Cerebrolysin, an injectable claimed to have nootropic properties,is produced by controlled enzymatic breakdown of pig brain pro-teins. Nowadays, it is only approved in Russia, China, and someother countries as a nootropic preparation. However, it can easilybe globally obtained through the Internet. The Internet-obtainedsample is composed of a large number of peptides, originatingfrom proteins, with some of them (like MBP) only being present inthe nervous system. The Mammalia database-compared sequencescorresponding to the filtered DDA and enhanced DDA spectra wereall linked to pig proteins which implicates that there is no evidencefor the presence of other mammalian proteins in the sample. Fur-ther, only two spectra could be assigned to Bacterian sequences.Nevertheless, after BLAST search or de novo sequencing of thesetwo spectra, the obtained sequences showed high similarity withpeptides from porcine collagen alpha-chain precursor and porcinealpha-tubulin. These findings support themanufacturer’s claim thatCerebrolysin is a pig-brain-derived product. A relatively high num-ber of MBP-derived peptides are found, which can be explainedby the fact that MBP is, next to proteolipid protein, the secondmostabundant protein (30%) in the myelin sheath (i.e., a highly abun-dant protein-lipid mixture surrounding the axons of nerve cells).[47]

However, it is remarkable that no proteolipid protein-derived pep-tides could be identified. This can be explained by its hydrophobicproperties, resulting in a loss during the Cerebrolysin production.[48]

Next to MBP, mainly peptides originating from tubulin alpha-1A, tu-bulin beta, and actin are found. Peptides from these proteins areidentified with the high resolution but also with the low resolutionmass analyzer, emphasizing the still valid, although limited role ofthis equipment in the first analytical phases of the characterizationof suspicious medicines. Full identification of the different compo-nents in this complex sample requires more high-end equipment.Especially, the ion mobility separation used in our characterizationhas a definite added value as was clearly demonstrated with thenumbers of (unique) identified peptides.The mechanism of action of Cerebrolysin is not completely

clarified, but the product may help the recovery after an acute is-chemic stroke, slow down the progression of vascular dementia,and shows nootropic effects that are similar to known neuro-trophic factors like BDNF, GDNF, NGF, and CNTF. However, wecould not identify any peptide originating from these proteins.Furthermore, we could not confirm the identification of frag-ments of enkephalins, orexin and galanin as reported byGromova et al.[17] If the mechanism of action of Cerebrolysin is


indeed similar to that of these neurotrophic factors, our resultsindicate that peptides from other proteins like MBP, actin, tubulin,or the other identified proteins without known nootropic proper-ties, are likely responsible for the reported effects. If so, the newactive peptides and their derived proteins can be labelled ascryptic peptide systems, i.e., proteins containing hidden func-tional peptide sequences.[49] These bioactive peptides are pro-duced in a controlled way with enzymes known as prohormoneconvertases or are formed upon protein degradation. Until now,no cryptides, i.e., peptides originating from a cryptic system, havebeen reported for the identified proteins. Nevertheless, peptideMBP 114–126 (FSWGAEGQKPGFG), also identified as one of thecomponents in Cerebrolysin, is being studied for its possible rolein the development of the autoimmune disease multiple sclerosis.Its mechanism of action in disease development and progressionis currently believed to be due to its weak MHC-antigen-T-cellreceptor (TCR) complex, and thus can this peptide not becategorized as a cryptide.[50] As MBP plays an important rolein the development of multiple sclerosis, it is hypothesizedthat fragments of MBP could possibly be useful as potentialtreatment for it.[51] Until now, different strategies have beentried as treatment for multiple sclerosis. Good results wereobtained with dirucotide, an MBP 82–98 (DENPVVHFFKNIVTPRT)synthetic peptide.[51] However, this investigational medicine failedduring phase III clinical trials in delaying the disease progression,as measured by the Expanded Disability Status Scale (EDSS) sosubsequent trials were cancelled.[51] Interestingly, an analogoussequence (MBP 85–97) was identified in the Cerebrolysin sample.Our findings about the composition can thus stimulate morebiomedical research in this field.

Conclusion

By LC-MS, we identified 638 unique peptides in an Internet-obtained Cerebrolysin sample which is claimed to havenootropic properties. No fragments of known nootropicproteins were identified and the reported biological effectsare thus likely originating from peptides from other precursorsthan BDNF, GDNF, NGF, and CNTF. MBP EGQKPGF-containingsequences in particular are highly abundant. The added valueof high resolution MS/MS – including ion mobility separation –

is clearly demonstrated by the number of identified peptidesand unique sequences compared to the other mass analyzersand acquisition techniques.



Drug Testing

and Analysis

Conflict of Interest

Johannes PC Vissers, Martin De Cecco and Jan Claereboudt areemployed by Waters Corporation, Milford, MA.

Acknowledgements

This research was funded by PhD grants from the Institute forthe Promotion of Innovation through Science and Technologyin Flanders (IWT-Vlaanderen) (Grant numbers 121512 to B.G.and 101529 to M.D.), the Special Research Fund (BOF) of GhentUniversity (Grant number 01J22510 to B.D.S. and E.W.), and theChina Scholarship Council (CSC to H.Y.).

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Peptide profiling of Internet-obtained Cerebrolysin using high performance liquid chromatography - electrospray ionization ion trap and ultra high performance liquid chromatography