A Proteomic-Based Approach for Detection of Chicken in Meat Mixes

Abstract

A proteomic-based method has been developed for the detection of chicken meat within mixed meat preparations. The procedure is robust and simple, comprising the extraction of myofibrillar proteins, enrichment of target proteins using OFFGEL isoelectric focusing, in-solution trypsin digestion of myosin light chain 3, and analysis of the generated peptides by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Using this approach, it was possible for example to detect 0.5% contaminating chicken in pork meat with high confidence. Quantitative detection of chicken meat was done by using AQUA stable isotope peptides made from the sequence of previously selected species-specific peptide biomarkers. Linearity was observed between the amount of the peptide biomarker and the amount of chicken present in the mixture; further independent replication is required now to validate the method. Apart from its simplicity, this approach has the advantage that it can be used effectively for the detection of both raw and cooked meat. The method is robust, reliable, and sensitive, representing a serious alternative to methods currently in use for these purposes. It is amenable to highly processed foods which can be particularly problematic, as the tertiary protein structure is often affected in processed food precluding immunoassays. In addition, this proteomic analysis will permit the determination of definitive discriminatory sequence, unlike the DNA PCR based methods used presently. The present article also demonstrates the translation of the technology to routine mass spectrometry equipment, making the methodology suitable for public analysts.

Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication

“…A more typical route is to use proteomic methods to identify various disparate species-dependent marker peptides, in which case the markers for different species possess no particular relationship with one another [8][9][10][11][12]14,19 . By contrast, we have selected proteins common to all species of interest up to species-dependent sequence variants 3 .…”

“…Species identification then normally proceeds via detection of disparate marker peptides, whereas in the CPCP approach species identification proceeds via detection of closely related peptides possessing typically one or two sequence differences. Finally, quantitation of proteins to estimate the percent by weight of one species in another might conventionally proceed via absolute quantitation of each protein separately based on known standards 7,14,15 . However using the CPCP method there is no need for calibration methods.…”

“…Other authors have used mass spectrometry to test for meat adulteration via peptide markers but from disparate proteins [8][9][10][11][12][13][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.…”

“…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 quantitation 14,15 .…”

Food Proteomics: Mapping Modifications

“…Proteomics also has proved to be useful for detecting chicken in mixed meat preparations. Sentandreu et al (2010) developed a method for the extraction of myofibrillar proteins and the subsequent enrichment of target proteins using OFFGEL isoelectric focusing, which were then identified by LC-MS/MS. Quantitative detection of chicken meat was achieved using AQUA stable isotope peptides and it was possible to detect contaminating chicken down to levels of 0.5%.…”