Arter Bestemmelse og kvantifisering i blandinger Bruke MRM massespektrometri av Peptider Applied til Meat Authentication
Summary
We present a protocol for identifying and quantifying the components in mixtures of species possessing similar proteins. Mass spectrometry detects peptides for identification, and gives relative quantitation by ratios of peak areas. As a tool food for fraud detection, the method can detect 1% horse in beef.
Abstract
We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. Central to the method is the identification of ‘corresponding proteins’ in the species of interest, in other words proteins that are nominally the same but possess species-specific sequence differences. When subject to proteolysis, corresponding proteins will give rise to some peptides which are likewise similar but with species-specific variants. These are ‘corresponding peptides’. Species-specific peptides can be used as markers for species determination, while pairs of corresponding peptides permit relative quantitation of two species in a mixture. The peptides are detected using multiple reaction monitoring (MRM) mass spectrometry, a highly specific technique that enables peptide-based species determination even in complex systems. In addition, the ratio of MRM peak areas deriving from corresponding peptides supports relative quantitation. Since corresponding proteins and peptides will, in the main, behave similarly in both processing and in experimental extraction and sample preparation, the relative quantitation should remain comparatively robust. In addition, this approach does not need the standards and calibrations required by absolute quantitation methods. The protocol is described in the context of red meats, which have convenient corresponding proteins in the form of their respective myoglobins. This application is relevant to food fraud detection: the method can detect 1% weight for weight of horse meat in beef. The corresponding protein, corresponding peptide (CPCP) relative quantitation using MRM peak area ratios gives good estimates of the weight for weight composition of a horse plus beef mixture.
Introduction
The European horse meat scandal of 2013, in which undeclared horse meat was found in a number of supermarket beef products1, highlights the need for testing methods capable of detecting and measuring food fraud in meat. Several technologies have been explored, especially enzyme-linked immunosorbent assay (ELISA) and DNA-based methods2. An alternative route, based on mass spectrometry, targets species-specific peptides which in turn arise from species-specific proteins. Here we outline one such peptide-based approach that offers both identification and relative quantitation of the adulterant species in a meat mixture3.
The protocol is framed in the context of red meats and the desire to determine the presence of one in another at the level of 1% by weight, the level considered by some to represent fraudulent food adulteration as opposed to contamination4. The method relies in the first instance on identifying a protein which is nominally ‘the same’ in all target meats. Myoglobin, the protein responsible for the red color of meat, is a good candidate since it is abundant, relatively heat tolerant and water soluble, and has been used for species determination of meat previously5,6. The myoglobins for beef (Bos Taurus), pork (Sus scrofa), horse (Equus caballus) and lamb (Ovis aries)3, for instance, are nominally the same, as required, but their sequences are not identical. Such groups of ‘similar but different’ proteins, like these four myoglobins, can conveniently be described as ‘corresponding proteins’. The sequence differences in these four myoglobins are species-specific: for example, the full myoglobin proteins for beef and horse, P02192 and P68082 respectively, each comprise 154 amino acids with 18 sequence differences between the two. Subject to proteolysis using trypsin these proteins produce two sets of peptides, some of which are identical, and some which show one or more species-specific amino acid differences: corresponding proteins therefore give rise to corresponding peptides.
The CPCP approach, therefore, seeks first to identify proteins from two or more species where these proteins exhibit limited species-specific sequence variants. These are corresponding proteins. Following proteolysis, corresponding proteins give rise to peptides, some of which likewise display species-specific sequence variants inherited from the parent protein. These are corresponding peptides. The CPCP approach can be used to compare levels of two corresponding proteins in a mixed species sample by monitoring the levels of corresponding peptides.
The natural technology for the detection of known peptides is multiple reaction monitoring mass spectrometry, or MRM-MS7. Species-specific peptides yield precursor ions, which along with their mass spectrometry fragment ions, are easily itemized in advance by software tools. These lists are then used to instruct the mass spectrometer to record only specific precursor plus fragment ion pairs, called transitions. A particular target peptide is therefore identified not only by its retention time in the chromatography preceding the mass spectrometer, but also by a set of transitions sharing a common precursor ion. This is a highly selective means of detecting known peptides that makes efficient use of the mass spectrometer resource.
Other authors have used mass spectrometry to test for meat adulteration via peptide markers but from disparate proteins8-14. Using the corresponding proteins, corresponding peptides (CPCP) scheme, however, means experimental conditions can be optimized, aiding identification of the species in the mixture from known species-specific transitions. In addition, corresponding proteins and peptides will generally behave similarly in the extraction, proteolysis and detection stages. Since transition peak areas are quantitative and reproducible, ratios of peak areas arising from pairs of corresponding peptides from different species provide a direct estimate of the relative quantities of two meats in a mixture. In contrast, more traditional quantitation routes exploit calibrations based on reference materials to establish absolute quantitation14,15.
Though the protocol is outlined in the context of myoglobin and meat, proteins other than myoglobin could be used for identification and relative quantitation via the CPCP strategy in meat mixtures, though potentially with modifications to the protocol. In addition the strategy is also applicable to binary mixtures of other species sharing one or more corresponding proteins.
The starting point for the protocol is purified ‘reference’ myoglobin, which for some species can be purchased but which for others must be prepared by conventional size-exclusion chromatography. The procedure for preparing reference myoglobin is not included in the protocol, but is described elsewhere3. Software tools16 are used to list candidate peptides and transitions arising from myoglobins of interest. Each reference myoglobin is subjected to proteolysis and the resultant peptides analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to discover which of the candidate precursor ions and transitions are most useful, and to determine the matching peptide retention times. The outcome of this stage is a revised list of target peptides with their transitions, suitable for species determination, and a list of CPCP pairs, suitable for relative quantitation. To test real meats, sample extractions are prepared then subjected to proteolysis to generate peptides both from myoglobin and other extraneous proteins. The myoglobin-based peptides are then monitored by LC-ESI-MS/MS based on their listed transitions. The species present in a mixture are identified by the transition peaks associated with marker peptides. Estimates of the relative amounts of two meats in a binary mixture are calculated using ratios of transition peak areas. A set of test mixtures of pairs of meats will allow the ratio of peak areas for a given pair of transitions to be checked and calibrated against actual mixtures.
Protocol
Representative Results
Discussion
Utvelgelsen av en passende målprotein er viktig. Et godt mål protein må ha tilsvarende former i arter av interesse, tilstrekkelig artsavhengige sekvensvariasjon, arter spesifisitet, og eksisterer i tilgjengelige mengder innenfor de organismer. For vurdering av blandinger som har gjennomgått behandlingen (for eksempel varmebehandling), et protein med en sekvens forholdsvis immun mot at behandling er ønskelig. Myoglobin er en god kandidat for rødt kjøtt, inkludert kokt rødt kjøtt, men er ikke den eneste muligheten. Når mål-proteinet er bestemt, er den mest kritiske del av protokollen er proteinet proteolyse. Et protein forskjellig fra myoglobin kan godt kreve en alternativ proteolyse protokollen.
Protokollen som beskrevet omfatter et segment basert på henvisning renset protein. Målet er å oppdage oppbevaring tidsvinduer og egnede forløper og fragmentioner. Dette segmentet er svært nyttig, men ikke avgjørende.
<pclass = "jove_content"> Selv om det tilsvarende peptid-parene fra to arter av interesse kan listes opp selv uten eksperiment, er det noen ganger slik at en sekvens forskjell har dramatiske konsekvenser for fordøyelsen profilen. For eksempel peptid par VLGFHG (biff) og ELGFQG (hest) gi en isolert kvantifisering resultat (manifest som en gradient mindre enn en i figur 2). Dette er fordi den sistnevnte peptid som oppstår fra en forholdsvis trykt KE spalting, forårsaker en under-estimat av nivået av hesten i blandingen. Tilsvarende peptider som starter med forskjellige aminosyrer er derfor best unngås. Ofte fragmenter fra to tilsvarende peptider har identiske aminosyresekvenser og veloppdragen, men dette er ikke alltid tilfelle, og må kontrolleres under metodeutvikling. Artsbestemmelse er mye mindre følsom for disse problemene enn relativ kvantifisering.Protokollen er påvist i fire rødt kjøtts 3. Andre kjøtt art kan inkluderes, selv om kvaliteten av overgangstoppform kan forringes hvis for mange peptider markør ko-eluerte, effektivt redusere oppholdstiden, og til slutt nedbryte relative kvantiteringsstandarder anslag. Forbedret instrumentering, som allerede er tilgjengelig, vil forbedre dette. Et beslektet problem er at ikke alle kjøtt har forskjellige myoglobins. For eksempel, hest, esel og sebra myoglobins er identiske og dermed strengt tatt metoden er bare i stand til å oppdage hest eller esel eller sebra i biff. I noen tilfeller, selv om myoglobins ikke er identiske, kan noen viktige peptider være. For eksempel, noen lam myoglobin-avledet markør peptider vises også i geit.
En komplikasjon som vender mot denne og andre proteinbaserte deteksjonsmetode er at proteinnivået må antas konstant over alle arter dersom protein eller peptid-nivåer er å likestille trivielt å nivåer av kjøtt i en blanding. For myoglobin og fire røde mspiser dette er ikke universelt sant. Nivåene generelt er artsavhengig, med svinekjøtt oppviser det laveste nivået av de fire. I tillegg varierer den myoglobin nivå med kjøtt og dyr snitt alder. Så selv om forholdene mellom overgangstopparealene kart på en pålitelig måte til prosenter av myoglobin, kartleggingen til forholdet mellom selve kjøttet er et anslag tegning på antagelser om sannsynlige kilder for kjøtt i blandingen.
Fremgangsmåten som er beskrevet i dette arbeidet skiller seg på flere måter fra andre publiserte bidrag. En mer typisk rute er å bruke proteomic metoder for å identifisere forskjellige uensartede artsspesifikke markør peptider, i hvilket tilfelle markører for forskjellige arter besitter ingen bestemt forhold til hverandre 8-12,14,19. Derimot, har vi valgt proteiner som er felles for alle arter av interesse opp til artsavhengige sekvensvarianter tre. Bortsett fra å være sentral i vår relative kvantifisering strategi, har dette den fordelen at prøvenforberedelse strategier kan optimaliseres. I tillegg kan slike korresponderende proteinene kan forventes å oppføre seg på samme måte, for eksempel ved ekstraksjon eller ved kommersiell behandling av prøver som matlaging eller hermetisering. Artsidentifikasjon deretter forløper normalt via detektering av ulike peptider markør, mens i de CPCP tilnærmingen arten identifikasjons fortsetter via detektering av nært beslektede peptider som innehar vanligvis en eller to sekvensforskjeller. Til slutt, kvantifisering av proteiner til å estimere vektprosent av en art i en annen kan på vanlig måte skje via absolutt mengdebestemmelse av hvert protein separat basert på kjente standarder 7,14,15. Men ved hjelp av CPCP metode er det ikke behov for kalibreringsmetoder. I stedet er relative nivåer estimert ved å sammenligne signalstyrkene til to tilsvarende peptider fra de to artene, utenom den absolutte måletrinnet fullstendig. Siden det endelige målet er en vektprosent av en art i another, en relativ kvantifisering, så CPCP er både mer direkte og enklere enn å sammenligne to absolutte kvantiteringsstandarder målinger. Disse funksjonene oversette til korte eksperimentelle ganger forventet å være om lag to timer ved hjelp av raffinerte protokoller, slik at teknikken er nyttig som en rask overvåking verktøy i riket av mat svindeldeteksjon.
Disclosures
The authors have nothing to disclose.
Acknowledgements
We acknowledge financial support from Institute of Food research BBSRC Core Strategic Grant funds, BBSRC Project BB/J004545/1.
Materials
| Uniprot database | www.uniprot.org | Freely accessible database of protein sequences | |
| Skyline software | www.skyline.gs.washington.edu | Free software to download that enables the creation of targeted methods for proteomic studies, peptide and fragment prediction | |
| Ammonium bicarbonate | Sigma-Aldrich Co Ltd, Gillingham, UK www.sigmaaldrich.com | O9830 | |
| Methanol, HPLC grade | Fisher Scientific, Loughoborough, UK www. fisher.co.uk | 10674922 | |
| Acetonitrile, HPLC grade | Fisher Scientific, Loughoborough, UK www. fisher.co.uk | 10010010 | |
| Urea | Sigma-Aldrich Co Ltd, Gillingham, UK www.sigmaaldrich.com | U5378 | |
| Trypsin(from bovine pancreas treated with TPCK) | Sigma-Aldrich Co Ltd, Gillingham, UK www.sigmaaldrich.com | T1426 | |
| Formic acid | Sigma-Aldrich Co Ltd, Gillingham, UK www.sigmaaldrich.com | F0507 | |
| Coomassie Plus Protein Assay Reagent | Thermo Fisher Scientific www.thermofisher.com | 1856210 | |
| Protein standard | Sigma-Aldrich Co Ltd, Gillingham, UK www.sigmaaldrich.com | P0914 | |
| Ultra Turrax homogeniser T25 | Fisher Scientific, Loughoborough, UK www. fisher.co.uk | 13190693 | |
| Edmund and Buhler KS10 lab shaker | |||
| Heraeus Fresco 17 Centrifuge | Thermo Fisher Scientific www.thermoscientific.com | 75002420 | |
| Vacuum centrifuge RC 1022 | Jouan | ||
| Plate Reader | |||
| Strata-X 33u polymeric reversed-phase cartridges 60 mg/3 ml tubes | Phenomenex, Macclesfield, UK | 8B-S100-UBJ | |
| 4000 QTrap triple-quadrupole mass spectrometer | AB Sciex, Warrington, UK www.sciex.com | ||
| 1200 rapid resolution LC system | Agilent, Stockport, UK | ||
| XB C18 reversed-phase capillary column (100 x 2.1mm, 2.6µ particle size) | Phenomenex, Macclesfield, UK www.phenomenex.com | ||
| Analyst 1.6.2 software | AB Sciex, Warrington, UK www.sciex.com | QTrap data acquisition and analysis, including peak area integration | |
| Autosampler vials |
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