Species-specific Peptide Markers Research Articles

Relative quantification of pork and beef in meat products using global and species-specific peptide markers for the authentication of meat composition

We used global and species-specific peptide markers for a relative quantitative determination of pork and beef in raw and processed meat products made of the two meat species. Four groups of products were prepared (i.e., minced raw meats, sausages, raw and fried burgers) in order to represent products with different extents of food processing. In each group, the products varied in the pork/beef proportions. All products were analysed by multiple reaction monitoring mass spectrometry (MRM–MS) for the presence/concentration of pork- and beef-specific peptide markers, as well as global markers – peptides widely distributed in muscle tissue. The combined MRM-MS analysis of pork-specific peptide HPGDFGADAQGAMSK, beef-specific peptide VLGFHG and global marker LFDLR offered the most reliable validation of declared pork/beef compositions across the whole range of meat products. Our work suggests that a simultaneous analysis of global and species-specific peptide markers can be used for composition authentication in commercial pork/beef products.

Mass spectrometry determination of potential species-specific peptide markers in commercial seasoning cubes

In recent years, issues related to food product authenticity have been alarming, particularly in meat-based products. Proteomic techniques in food authentication and their applications in determining quality and safety issues of ingredients have been progressively developed by researchers. The complexity of matrices in commercial seasoning cubes is one of the main concerns in developing a robust authentication method. Hence, the present study focuses on the development of a protein extraction method for commercial seasoning cubes and to determine potential species-specific peptide markers in commercial seasoning cubes. The efficacy of solvent extraction was determined using Bradford assays, while the electrophoretic profiles of commercial seasoning cubes were visualized via Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The determination of potential species-specific peptide markers in commercial seasoning cubes was carried out via Liquid Chromatography (LC)-Orbitrap Fusion-Mass spectrometry (MS/MS) analysis (Thermo Fisher Scientific). In this study, the urea-thiourea solvent was ideal in extracting protein from commercial seasoning cubes as it produces a homogenous solution upon mixing, suggesting the diffusibility of solutes that is essential for continuing analysis. Moreover, a bovine-specific peptide marker ‘VGGHAAEYGAEALER’ and two porcine-specific peptide markers; ‘VGGQAGAHGAEALER’ and ‘EAVLGLWGK’ were discovered. The methodology developed in this study was successful in identifying species-specific peptide markers in seasoning cubes and can be used as a reference for validating the authenticity of food products, especially commercial seasonings.

A quantitative strategy of ultrasound-assisted digestion combined UPLC-MS/MS for rapid identifying species-specific peptide markers in the application of food gelatin authentication

Gelatins are important and frequently-used food additives, but the quality control of gelatins is always an intractable problem in routine analysis. In this work, a new strategy for simultaneously rapid identification and quantification of gelatins from various species using ultrasound assisted digestion-UPLC-MS/MS was described. Fourteen peptide markers were used for authenticity of gelatins by the presence or absence of these species-specific peptides. In the meantime, adulteration ratios could be calculated based on the peak area of different species peptide markers. With 10 g/L trypsin and ultrasound application, digestion time could be decreased from >12 h (traditional method) to 5 min, and the determination of animal gelatins could be achieved in 20 min with a single analysis run. Twenty-five commercial gelatin samples were screened, among which two deer-hide gelatin samples were adulterated with 900 g/kg and 265 g/kg cattle-hide gelatin (CHG); two donkey-hide gelatin (ACC) samples were adulterated with 66 g/kg and 381 g/kg horse-hide gelatin; and one ACC sample was adulterated with 786 g/kg CHG. This strategy provides an efficient and sensitive authentication and traceability of gelatin-containing products, and could also be applied to processed meat or protein food.

Multispecies Identification of Oilseed- and Meat-Specific Proteins and Heat-Stable Peptide Markers in Food Products.

Consumer demand for both plant products and meat products enriched with plant raw materials is constantly increasing. Therefore, new versatile and reliable methods are needed to find and combat fraudulent practices in processed foods. The objective of this study was to identify oilseed species-specific peptide markers and meat-specific markers that were resistant to processing, for multispecies authentication of different meat and vegan food products using the proteomic LC-MS/MS method. To assess the limit of detection (LOD) for hemp proteins, cooked meatballs consisting of three meat species and hemp cake at a final concentration of up to 7.4% were examined. Hemp addition at a low concentration of below 1% was detected. The LOD for edestin subunits and albumin was 0.9% (w/w), whereas for 7S vicilin-like protein it was 4.2% (w/w). Specific heat-stable peptides unique to hemp seeds, flaxseed, nigella, pumpkin, sesame, and sunflower seeds, as well as guinea fowl, rabbit, pork, and chicken meat, were detected in different meat and vegan foods. Most of the oilseed-specific peptides were identified as processing-resistant markers belonging to 11S globulin subunits, namely conlinin, edestin, helianthinin, pumpkin vicilin-like or late embryogenesis proteins, and sesame legumin-like as well as 2S albumins and oleosin isoforms or selected enzymic proteins.

Identification of species-specific peptide markers in cold-pressed oils

In recent years, cold-pressed vegetable oils have become very popular on the global market. Therefore, new versatile methods with high sensitivity and specificity are needed to find and combat fraudulent practices. The objective of this study was to identify oilseed species-specific peptide markers, using proteomic techniques, for authentication of 10 cold-pressed oils. In total, over 380 proteins and 1050 peptides were detected in the samples. Among those peptides, 92 were found to be species-specific and unique to coconut, evening primrose, flax, hemp, milk thistle, nigella, pumpkin, rapeseed, sesame, and sunflower oilseed species. Most of the specific peptides were released from major seed storage proteins (11 globulins, 2S albumins), and oleosins. Additionally, the presence of allergenic proteins in the cold-pressed oils, including pumpkin Cuc ma 5, sunflower Hel a 3, and six sesame allergens (Ses i 1, Ses i 2, Ses i 3, Ses i 4, Ses i 6, and Ses i 7) was confirmed in this study. This study provides novel information on specific peptides that will help to monitor and verify the declared composition of cold-pressed oil as well as the presence of food allergens. This study can be useful in the era of widely used unlawful practices.

Proteomic analysis of oilseed cake: a comparative study of species-specific proteins and peptides extracted from ten seed species.

In recent years there has been a visible trend among consumers to move away from consuming meat in favor of plant products. Meat producers have therefore been trying to meet the expectations of consumers by introducing new products to the food market with a greater proportion of plant ingredients. Meat products are enriched not only by the addition of vegetable oils but also by ground or whole oilseeds or their preparation. In this study, we present in-solution tryptic digestion and an ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS)-based proteomics approach to investigate specific proteins and peptides of ten oilseed cakes, by-products of cold pressing oil from coconut, evening primrose, hemp, flax, milk thistle, nigella, pumpkin, rapeseed, sesame, and sunflower seeds, for authentication purposes. We identified a total of 229 unique oilseed proteins. The number of specific proteins varied depending on the sample, from 4 to 48 in evening primrose and sesame. Moreover, we identified approximately 440 oilseed unique peptides in the cakes of all the analyzed oilseeds; the largest amounts were found in sesame (107 peptides), sunflower (100), pumpkin, hemp (42), rapeseed (36), and flax cake (35 peptides). We provide novel information on unique / species-specific peptide markers that will extend the scope of testing the authenticity of a wide range of foods. The results of this peptide discovery experiment may further contribute to the development of targeted methods for the detection and quantification of oilseed proteins in processed foods, and thus to the improvement of food quality. © 2020 Society of Chemical Industry.

Identification and absolute quantification of animal blood products by peptide markers using an UPLC–MS/MS method

Animal blood products, also known as "liquid meat" or “blood tofu”, are rich in many nutrients, consumed in large quantities, and plagued by adulteration. In this study, a qualitative and absolute quantitative method for blood tofu of different animal species, including pig, bovine, sheep, chicken, and duck was established. Species-specific peptide markers were screened using Proteome Discoverer software after data analysis by high-resolution mass spectrometry. Then, calibration and quantitative analysis were used to identify peptides which could be used for accurate quantification. Finally, three peptides of each species were selected for accurate qualitative and quantitative analyses. The linear correlation coefficients were all greater than 0.995, and the deviation of the quantitative results was 80–120%. The method can directly quantify the accurate blood content in blood tofu.

LIQUID CHROMATOGRAPHY–MASS SPECTROMETRY BOTTOM‐UP PROTEOMIC METHODS IN ANIMAL SPECIES ANALYSIS OF PROCESSED MEAT FOR FOOD AUTHENTICATION AND THE DETECTION OF ADULTERATIONS

This review offers an overview of the current status and the most recent advances in liquid chromatography-mass spectrometry (LC-MS) techniques with both high-resolution and low-resolution tandem mass analyzers applied to the identification and detection of heat-stable species-specific peptide markers of meat in highly processed food products. We present sets of myofibrillar and sarcoplasmic proteins, which turned out to be the source of 105 heat-stable peptides, detectable in processed meat using LC-MS/MS. A list of heat-stable species-specific peptides was compiled for eleven types of white and red meat including chicken, duck, goose, turkey, pork, beef, lamb, rabbit, buffalo, deer, and horse meat, which can be used as markers for meat authentication. Among the 105 peptides, 57 were verified by multiple reaction monitoring, enabling identification of each species with high specificity and selectivity. The most described and monitored species by LC-MS/MS so far are chicken and pork with 26 confirmed heat-stable peptide markers for each meat. In thermally processed samples, myosin, myoglobin, hemoglobin, l-lactase dehydrogenase A and β-enolase are the main protein sources of heat-stable markers. © 2019 John Wiley & Sons Ltd. Mass Spec Rev.

Quantitative identification of muscular tissue by the means of protototic peptides using the multiple reaction monitoring method

One of the main problems of modern production of meat products is the quality of raw meat, which depends on various factors including genetic components, conditions of transportation, production and processing. The most important components of meat are proteins, the total content, structure and functional state of which in this complex biological system with many interacting components is constantly changing. In order to study the interspecific and intraspecific features of meat proteins, their transformations in the process of maturation and processing requires modern analytical technologies based on the systematic approach to the analysis. Proteomics, as a methodology for studying proteins in a certain system and at a certain time, opens wide opportunities in this direction, allowing to identify and develop accurate analytical methods for searching biomarkers and identifying unfair practices. Given the high added value and multicomponent composition, finished meat products are among the most susceptible to adulteration. Current paper presents a technique of high-performance liquid chromatography with mass spectrometric detector ( HPLC-MS ) adapted for the detection and quantification of two different types of meat (beef and pork) in a complex biological matrix such as structureless minced meat. After the protein isolation and trypsin cleavage, species-specific peptide markers were selected for each animal species for the quantification. The technique was tested on model samples of a mixture of muscle tissue of the two species of animals. A good sensitivity was established with the possibility of quantitative determination of muscle tissue of each animal species using special calibration graphs. The developed technique can find wide application at the supervising organizations aimed at counteracting the discrepancies in the hidden replacement of types of meat by cheaper or low-grade raw materials. Keywords : Biomarker, prototypical peptides, HPLC-MS, species identification (Russian) DOI: http://dx.doi.org/10.15826/analitika.2019.23.4.012 D.V. Khvostov 1 , N.L. Vostrikova 1 , A.V. Zherdev 2 , E.A. Zvereva 2 , A.A. Kurzova 1 1 V.M. Gorbatov Federal Research Center for Food Systems of Russian Academy of Sciences , 109316 Moscow, Russian Federation 2 Federal State Institution «Federal Research Centre «Fundamentals of Biotechnology» of the Russian Academy of Sciences», Leninsky prospect, 33, build. 2, 119071 Moscow, Russian Federation

Chemometrics-Assisted Shotgun Proteomics for Establishment of Potential Peptide Markers of Non-Halal Pork (Sus scrofa) among Halal Beef and Chicken

Authentication of meat is a global concern owing to meat adulteration with pork; however, no study, to date, has established a method to identify species-specific peptide markers for ascertaining adulterated meat. The present study aimed to describe chemometrics-assisted shotgun proteomics, which has recently been reported to establish potential peptide markers for non-Halal pork among Halal beef and chicken meat. To identify the potential peptide markers through peptide mass fingerprinting (PMF) analysis, chemometric analysis comprised principal component analysis and orthogonal partial least square-discriminant analysis, both of which proved statistically useful. Subsequently, through targeted tandem liquid chromatography-mass spectrometry (LC-MS) analysis, the primary structure of the identified peptide markers was revealed using the de novo identification approach. A decoy, randomised and concatenated database search program comprising MS-Fit and MS-Tag was used for PMF and targeted tandem LC-MS analysis, respectively. Results obtained using both PMF and targeted tandem LC-MS analysis complemented one another, indicating that these methods can yield consistent results to identify peptide markers together with chemometric analysis.

A Genoproteomic Approach to Detect Peptide Markers of Bacterial Respiratory Pathogens.

Rapid identification of respiratory pathogens may facilitate targeted antimicrobial therapy. Direct identification of bacteria in bronchoalveolar lavage (BAL) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is confounded by interfering substances. We describe a method to identify unique peptide markers of 5 gram-negative bacteria by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for direct pathogen identification in BAL. In silico translation and digestion were performed on 14-25 whole genomes representing strains of Acinetobacter baumannii, Moraxella catarrhalis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Klebsiella pneumoniae. Peptides constituting theoretical core peptidomes in each were identified. Rapid tryptic digestion was performed; peptides were analyzed by LC-MS/MS and compared with the theoretical core peptidomes. High-confidence core peptides (false discovery rate <1%) were identified and analyzed with the lowest common ancestor search to yield potential species-specific peptide markers. The species specificity of each peptide was verified with protein BLAST. Further, 1 or 2 pathogens were serially diluted into pooled inflamed BAL, and a targeted LC-MS/MS assay was used to detect 25 peptides simultaneously. Five unique peptides with the highest abundance for each pathogen distinguished these pathogens with varied detection sensitivities. Peptide markers for A. baumannii and P. aeruginosa, when spiked simultaneously into inflamed BAL, were detected with as few as 3.6 (0.2) × 103 and 2.2 (0.6) × 103 colony-forming units, respectively, by targeted LC-MS/MS. This proof-of-concept study shows the feasibility of identifying unique peptides in BAL for 5 gram-negative bacterial pathogens, and it may provide a novel approach for rapid direct identification of bacterial pathogens in BAL.

Label-free quantification of meat proteins for evaluation of species composition of processed meat products

This study evaluated the application of label-free, relative quantification of highly processed meat proteins to authenticate processed meat products. Quantitation was based on proteins for which heat-stable and species-specific peptide markers had been detected. Using nano-LC-QTOF-MS/MS, 20 new, heat-stable peptide markers unique to chicken, duck and goose were identified, with significant scores. The method enabled detection of 1%(w/w) of chicken and 1%(w/w) pork in a mixture of the meat of three species, as well as 0.8%(w/w) beef proteins in commercial poultry frankfurters. The proteins’ abundance was computed using a spectral matching technique, termed the absolute protein expression (APEX) proteomics tool. This method includes a correction factor for each protein, based on the peptide MS detection probabilities, which are influenced by the physicochemical properties of the peptide. Considerable differences in abundance of myofibrillar and sarcoplasmic proteins were observed between samples and illegal proportions of ingredients were discovered.

Rapid detection of peptide markers for authentication purposes in raw and cooked meat using ambient liquid extraction surface analysis mass spectrometry.

In this Article, our previously developed ambient LESA-MS methodology is implemented to analyze five types of thermally treated meat species, namely, beef, pork, horse, chicken, and turkey meat, to select and identify heat-stable and species-specific peptide markers. In-solution tryptic digests of cooked meats were deposited onto a polymer surface, followed by LESA-MS analysis and evaluation using multivariate data analysis and tandem electrospray MS. The five types of cooked meat were clearly discriminated using principal component analysis and orthogonal partial least-squares discriminant analysis. 23 heat stable peptide markers unique to species and muscle protein were identified following data-dependent tandem LESA-MS analysis. Surface extraction and direct ambient MS analysis of mixtures of cooked meat species was performed for the first time and enabled detection of 10% (w/w) of pork, horse, and turkey meat and 5% (w/w) of chicken meat in beef, using the developed LESA-MS/MS analysis. The study shows, for the first time, that ambient LESA-MS methodology displays specificity sufficient to be implemented effectively for the analysis of processed and complex peptide digests. The proposed approach is much faster and simpler than other measurement tools for meat speciation; it has potential for application in other areas of meat science or food production.