14 2/14 eFOOD-Lab international Quality Management In addition, minor sequence differences as observed for closely related species are often overseen. Progress in the field of high resolution mass spectrometry in recent years nowadays allows the comprehensive characterization of complex proteomes. However, separation of complex protein mixtures using standard chromatography is still rather poor. Mass spectrometry in proteomics is therefore in general performed after enzymatic (mainly tryptic) digest of proteins to peptides which can be efficiently separated using reversed phase chromatography. In addition, fragmentation pattern of peptides in mass spectrometry are nicely predictable which allows peptide sequencing with high confidence. This approach is also termed “bottom-up” proteomics as the sequence information of proteins of interest is built up “from the bottom” (namely the analyzed peptides). Depending on the sample, up to 50,000 peptides are currently identified and sequenced in a single run using Bottom-up proteomics on state-of-the-art high resolution mass spectrometers (HRMS). This allows a highly parallel search for species-specific polymorphisms. For the Identification of biomarker peptides from pork (Sus scrofa) and horse (Equus caballus) we identified about 5,500 peptides per species after extraction of the myofibrillar and sarcoplasmic fraction of various food-relevant mammalian species and searched biomarker peptides by database search, de novo sequencing and comparison of experimental datasets 3. Peptides identified as species-specific need to be further validated concerning their usability in analytical methods. As an example, sequences containing methionine and cysteine should be avoided as these amino acids are highly prone to oxidation. For highest sensitivity, targeted proteomics on Triple-quadrupole or QTrap-instruments using multiple reaction monitoring (MRM) is most promising. After identification of species-specific peptides using HRMS often 50-100 biomarker peptide candidates are used for the first rounds of MRM method development and for all of these peptides MRM transitions need to be optimized to identify the most promising biomarkers. To this end, software-based optimization protocols are used, which allow method development for numerous peptides (and other analytes) in parallel without the need of purified peptide standards. However, peptides that are specific in HRMS are not automatically specific under MRM conditions as the reduced mass resolution leads to increasing matrix interferences. During method development for our HPLC-MS method for the detection of horse and pork we used approximately 70 potential biomarker peptides per species identified by HRMS and optimized about 500 MRM transitions on a AB Sciex QTtrap 5500. Specificity and sensitivity of peptide candidates was assessed experimentally against relevant mammalian Figure 4: Principle of the MRM3 technique species, resulting in the identification of the 10 most sensitive and specific peptides. For the overview of the workflow see Fig. 2. Further increase in sensitivity can be achieved on QTrap instruments using the MRM3 experiments (Fig. 3). In this secondary MRM experiment, the daughter ion of a MRM transition is fragmented to a “granddaughter ion” in the linear ion trap and detected (the principle is given in Fig. 4). Another possibility is the use of Micro- LC systems which, by reduction of flow rate and enhanced ionization efficiency, show advantages concerning throughput and sensitivity. The combination of MRM3 and Micro- LC resulted in a limit of detection of 0.1 % horse or pork in a beef matrix 3. Currently we optimize this method for the analysis of highly processed food samples with similar sensitivities and develop a multi-species method for the simultaneous detection of various food-relevant species. Conclusion The rapid progress in the field of mass spectormetry now allows for the detection of hundreds of proteins in complex matrices such as food. In addition, sensitive triple-quadrupole instruments are widely distributed in routine laboratories and can be used for targeted proteomics. For food analysis targeted proteomics is especially interesting for allergen detection and species authentication. However, the identification of species-specific peptides is still demanding and needs to be performed on HRMS instruments. In a pilot study we have therefore developed a detection method for horse and pork using MRM-based methods that can be directly applied in routine laboratories as an alternative to PCR and ELI SA methods. MRM-based methods are promising alternatives compared to established techniques especially for the analysis of processed samples and the simultaneous detection of different species in a single run. Literature 1. Pandey, A., Mann, M. (2000) Nature, 450, 837-846. 2. Castellana, N., Bafna, V. (2010) J Proteomics, 73, 2124-2135. 3. von Bargen, C., Dojahn, J., Waidelich, D., Humpf, H.-U., Brockmeyer, J. (2013) J Agric Food Chem, 61, 11986-11994.
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