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.
合适的靶蛋白的选择是重要的。一个好的目标蛋白质需要在目标物质相应的形式,充分物种依赖性序列变异,种属特异性,与生物体内存在着大量访问。评估已经历处理混合物(例如,热处理),具有相对地不受该处理的序列的蛋白质是理想的。肌红蛋白是红肉,包括熟食红肉一个很好的候选人,但并不是唯一的可能性。一旦目标蛋白质决定,该协议的最关键的部分是蛋白质水解。从不同的肌红蛋白的蛋白质很可能需要一个替代蛋白水解协议。
如所描述的协议包括基于参考纯化的蛋白质的片段。这旨在发现保留时间窗口和合适的前体和碎片离子。这部分是非常有益的,但不是必需的。
<p类=“jove_content”>尽管来自两个物种的兴趣相应肽对即使不实验中列出,有时,一个序列差异对消化轮廓严重后果的情况。例如,所述肽对VLGFHG(牛肉)和ELGFQG(马)得到异常的定量结果(表现为一个梯度小于一在图2中)。这是因为,后面的肽源于相对抑制KE裂解,引起马的混合物中的水平的低估。因此最好的开始与不同的氨基酸相应的肽被避免。常来自两个对应的肽片段具有相同的氨基酸序列,并表现良好,但是这并非总是如此,需要方法开发期间进行检查。种属鉴定是敏感的这些问题比相对定量少得多。该协议已被证明四红肉第3条 。附加肉种类可以包括,但过渡峰形的质量可能如果太多标记肽共洗脱恶化,有效地减少了停留时间和最终降解相对定量估计。改进的仪器,已经上市,将改善这一点。一个相关的问题是,并非所有的肉都有不同myoglobins。例如,马,驴和斑马myoglobins是相同的,因此严格来说该方法只能够在牛肉检测马,驴或斑马。在某些情况下,即使myoglobins不相同,一些关键的肽即可。例如,一些羊肉肌红蛋白衍生肽标记也出现在山羊。
面对此以及任何其他基于蛋白质的定量方法的复杂性在于,在蛋白质水平必须假定在所有物种恒定如果蛋白质或肽水平是平凡等同于混合物中的肉类的水平。对于肌红蛋白和四个红M吃这不是普遍如此。在一般的水平是物种依赖性的,猪肉呈现四个的最低水平。此外,肌红蛋白水平分割肉和动物的年龄而异。所以虽然过渡峰面积的比率可靠地映射到肌红蛋白的比率时,映射到实际的肉类的比率是对有关在该混合物的肉类的可能的来源的假设的估计图。
在这项工作中所概述的方法的不同之处的一些其他发表的贡献的方式。更典型的途径是使用蛋白质组学方法来识别各种不同的物种依赖性标记的肽,在这种情况下,对于不同物种的标记物具有彼此8-12,14,19没有特别的关系。相比之下,我们选择共同所有感兴趣的物种的蛋白质多达物种依赖性序列变体3。除了是中央到我们的相对定量的策略,这具有的优点是样品制备策略可以被优化。此外,这种相应的蛋白质可以预料到的行为类似,例如,在提取或样品如烹调或罐头的商业加工。种属鉴定,然后通过检测不同标记肽的正常进行,而通过检测通常拥有一个或两个序列的差异密切相关肽的CPCP办法物种鉴定的收益。最后,蛋白的定量在另一个重量的一种物种的估计的百分比通过分别基于已知标准7,14,15各蛋白质的绝对定量可能常规进行。然而,使用CPCP方法也没有必要进行校准的方法。相反,相对水平通过比较来自两个物种两个相应肽的信号强度,完全绕过绝对测量载台估计。由于最终目标是重量在ANO一个物种的百分比疗法,一个相对定量,则CPCP既更直接和超过比较两个绝对定量测量更简单。这些特点转化为短的实验时间,预计将用精致的协议,大约两小时,使得该技术可用作食品欺诈检测领域快速的监视工具。
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 |