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.
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.
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.
Udvælgelsen af et egnet target protein er vigtig. En god målprotein skal have tilsvarende former i arter af interesse, tilstrækkelig arter afhængige sekvensvariation, artsspecificitet, og findes i tilgængelige mængder inden for de organismer. For at vurdere blandinger som er behandlet (f.eks varmebehandling), et protein med en sekvens relativt immune over for denne behandling er ønskelig. Myoglobin er en god kandidat til rødt kød, herunder kogt rødt kød, men er ikke den eneste mulighed. Når målproteinet bestemmes, den mest kritiske del af protokollen er det protein proteolyse. Et protein forskelligt fra myoglobin kan meget vel kræve en alternativ proteolyse protokol.
Protokollen som beskrevet indeholder et segment baseret på henvisning renset protein. Dette har til formål at opdage fastholdelse tid vinduer og egnede prækursorer og fragment-ioner. Dette segment er meget nyttigt, men ikke afgørende.
<pclass = "jove_content"> Selvom tilsvarende peptid par fra to arter af interesse kan listet selv uden eksperiment, er det nogle gange sådan, at en sekvens forskel har dramatiske konsekvenser for fordøjelsen profil. For eksempel peptidet pair VLGFHG (oksekød) og ELGFQG (hest) giver en anomal kvantificering resultat (åbenbart som en gradient mindre end en i figur 2). Dette er fordi sidstnævnte peptid skyldes en forholdsvis undertrykt KE spaltning, der forårsager en undervurdering af niveauet af hesten i blandingen. Tilsvarende peptider starter med forskellige aminosyrer er derfor bedst undgås. Ofte fragmenterne fra to tilsvarende peptider har identiske aminosyresekvenser og er godt behaved, men dette er ikke altid tilfældet, og skal kontrolleres under udvikling metode. Arter identifikation er langt mindre følsomme over for disse spørgsmål end relativ kvantificering.Protokollen er blevet påvist for fire rødt køds 3. Yderligere kød arter kan indbefattes, selvom kvaliteten af overgangen topform kan forringes, hvis for mange markør peptider co-eluerer, effektivt at reducere opholdstiden og i sidste ende nedbrydning relative kvantificering skøn. Forbedret instrumentering, der allerede findes, vil forbedre dette. Et beslægtet problem er, at ikke alle kød har forskellige myoglobiner. For eksempel, hest, æsel og zebra myoglobiner er identiske og dermed strengt taget metoden er kun i stand til at detektere hest eller æsel eller zebra i oksekød. I nogle tilfælde, selv om myoglobiner ikke er identiske, kan nogle af de vigtigste peptider være. For eksempel vises nogle lam myoglobin-afledte markør peptider også i ged.
En komplikation overfor denne og andre protein-baserede kvantificering fremgangsmåde er, at proteinniveauet må antages konstant over alle arter, hvis proteinet eller peptidet niveauer er at sidestille trivielt til niveauer på kød i en blanding. For myoglobin og fire røde mæder dette ikke er universelt sandt. Niveauerne i almindelighed er arter afhængige, med svinekød udviser det laveste niveau af de fire. Derudover myoglobin niveauet varierer med udskårne kødstykke og dyrs alder. Så selv om forhold mellem overgangen toparealer kort pålideligt til forhold mellem myoglobin, kortlægning til forholdet mellem de faktiske kød er et skøn tegning på formodninger om sandsynlige kilder til kød i blandingen.
Strategien i dette arbejde adskiller sig på flere måder fra andre offentliggjorte bidrag. En mere typisk vej er at bruge proteom metoder til at identificere forskellige uensartede arter-afhængig markør peptider, i hvilket tilfælde de markører for forskellige arter besidder nogen særlig relation med hinanden 8-12,14,19. Derimod har vi udvalgt proteiner fælles for alle arter af interesse op til arter afhængige sekvens varianter 3. Ud over at være central for vores relative kvantificering strategi har dette den fordel, at prøvenstrategier forberedelse kan optimeres. Desuden kan sådanne tilsvarende proteiner forventes at opføre sig på samme måde, for eksempel ved udvindingen eller i kommerciel behandling af prøver såsom madlavning eller konserves. Arter identifikation derefter normalt forløber via påvisning af forskellige markør-peptider, mens der i de CPCP tilgang identifikation arter provenu via påvisning af nært beslægtede peptider besidder typisk en eller to sekvens forskelle. Endelig kvantificering af proteiner til at estimere den vægtprocent af én art i en anden kan konventionelt forløbe via absolut kvantificering af hvert protein separat baseret på kendte standarder 7,14,15. Men ved hjælp af CPCP metode er der ikke behov for kalibreringsmetoder. I stedet er relative niveauer anslås ved sammenligning signalstyrker to tilsvarende peptider fra de to arter, uden om absolut måling fase helt. Da det endelige mål er en vægtprocent af en art i another, en relativ kvantificering, så CPCP er både mere direkte og enklere end at sammenligne to absolutte kvantificeringsfremgangsmåder målinger. Disse funktioner omsættes til korte eksperimentelle gange, forventede at være cirka to timers hjælp raffinerede protokoller, hvilket gør teknikken anvendelig som en hurtig overvågning værktøj i realm af afsløring af svindel mad.
The authors have nothing to disclose.
We acknowledge financial support from Institute of Food research BBSRC Core Strategic Grant funds, BBSRC Project BB/J004545/1.
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 |