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Rapid analysis of multiple food allergens is required to confirm the appropriateness of food allergen labelling in processed foods. This study aimed to develop a rapid and reliable method to simultaneously detect trace amounts of seven food allergenic proteins (wheat, buckwheat, milk, egg, crustacean, peanut, and walnut) in processed foods using LC-MS/MS. Suspension-trapping (S-Trap) columns and on-line automated solid-phase extraction were used to improve the complex and time-consuming pretreatment process previously required for allergen analysis using LC-MS/MS. The developed method enabled the simultaneous detection of selected marker peptides for specific proteins derived from seven food ingredients in five types of incurred samples amended with trace amounts of allergenic proteins. The limit of detection values of the method for each protein were estimated to be

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Donkey-hide gelatin, also called Ejiao (colla corii asini), is commonly used as a food health supplement and valuable Chinese medicine. Its growing popular demand and short supply make it a target for fraud, and many other animal gelatins can be found as adulterants. Authentication remains a quality concern. Peptide markers were developed by searching the protein database. However, donkeys and horses share the same database, and there is no specific marker for donkeys. Here, solutions are sought following a database-independent strategy. The peptide profiles of authentic samples of different animal gelatins were compared using LC-QTOF-MS/MS. Fourteen specific markers, including four donkey-specific, one horse-specific, three cattle-specific, and six pig-specific peptides, were successfully found. As these donkey-specific peptides are not included in the current proteomics database, their sequences were determined by de novo sequencing. A quantitative LC-QQQ multiple reaction monitoring (MRM) method was further developed to achieve highly sensitive and selective analysis. The specificity and applicability of these markers were confirmed by testing multiple authentic samples and 110 batches of commercial Ejiao products, 57 of which were found to be unqualified. These results suggest that these markers are specific and accurate for authentication purposes. [ABSTRACT FROM AUTHOR]

Rapid analysis of multiple food allergens is required to confirm the appropriateness of food allergen labelling in processed foods. This study aimed to develop a rapid and reliable method to simultaneously detect trace amounts of seven food allergenic proteins (wheat, buckwheat, milk, egg, crustacean, peanut, and walnut) in processed foods using LC-MS/MS. Suspension-trapping (S-Trap) columns and on-line automated solid-phase extraction were used to improve the complex and time-consuming pretreatment process previously required for allergen analysis using LC-MS/MS. The developed method enabled the simultaneous detection of selected marker peptides for specific proteins derived from seven food ingredients in five types of incurred samples amended with trace amounts of allergenic proteins. The limit of detection values of the method for each protein were estimated to be <1 mg/kg. The developed analytical approach is considered an effective screening method for confirming food allergen labelling on a wide range of processed foods., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier Ltd.)

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Donkey-hide gelatin, also called Ejiao (colla corii asini), is commonly used as a food health supplement and valuable Chinese medicine. Its growing popular demand and short supply make it a target for fraud, and many other animal gelatins can be found as adulterants. Authentication remains a quality concern. Peptide markers were developed by searching the protein database. However, donkeys and horses share the same database, and there is no specific marker for donkeys. Here, solutions are sought following a database-independent strategy. The peptide profiles of authentic samples of different animal gelatins were compared using LC-QTOF-MS/MS. Fourteen specific markers, including four donkey-specific, one horse-specific, three cattle-specific, and six pig-specific peptides, were successfully found. As these donkey-specific peptides are not included in the current proteomics database, their sequences were determined by de novo sequencing. A quantitative LC-QQQ multiple reaction monitoring (MRM) method was further developed to achieve highly sensitive and selective analysis. The specificity and applicability of these markers were confirmed by testing multiple authentic samples and 110 batches of commercial Ejiao products, 57 of which were found to be unqualified. These results suggest that these markers are specific and accurate for authentication purposes.

Gelatine is one of the components commonly used in food, cosmetics and pharmaceutical products due to its gelling properties. The most commonly used gelatines in those products are porcine and bovine gelatines. Unclear labelling and information regarding the actual sources of gelatines in products have become the main concern among societies in terms of religion and health aspects. Porcine gelatine (PG) is prohibited to be consumed by Muslim and Jewish and considered non-halal (and non-kosher) following some scholars of thought. While bovine gelatine (BG) is associated with certain diseases of bovine spongiform encephalopathy, as a consequence, there is a need to develop reliable methods for identifying gelatine sources in the products. This review highlighted some analytical methods including physico-chemical methods as well as biological methods along with advantage and disadvantage for differentiation of gelatines intended to halal authentication studies. Key Findings and Conclusions: Some analytical methods are used for screening PG and BG using physico-chemical properties including chemical precipitation, functional groups (FTIR spectroscopy), amino acid composition (liquid chromatography), detection and quantification of DNA (real-time polymerase chain reaction/RT-PCR), molecular weight distribution (electrophoresis), and protein (Enzyme-linked immunosorbent assay, ELISA). These methods are confirmed by identification of peptide markers which are specific for PG and BG using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and DNA based method using polymerase chain reaction. • Porcine gelatine is widely used in food, pharmaceutical, and cosmetics products. • Porcine gelatine is prohibited especially for Muslim because it is categorized as non-halal substance. • Physico-chemical methods for porcine gelatine detection including FTIR, high performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry. • Biological methods such as real-time PCR offer selectivity and sensitivity for porcine gelatin identification. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

[Display omitted] • Peptides derived from G3P, ENOB, and KCRM were associated with increase in redness and yellowness. • The MYG, ENOB, HBA, PGK1, and TPIS peptides were closely related to tenderization of beef. • Peptide fragments can be indicators of fresh and aged meat quality characteristics. Peptides in fresh and aged beef strip loin (M. longissimus lumborum) and tenderloin (M. psoas major) were quantified to investigate the relationship between proteolysis-induced peptides and beef quality characteristics. A total of 409 and 450 peptides were quantified from strip loin and tenderloin, respectively, and found to be significantly correlated to beef quality characteristics. Changes in redness and yellowness were significantly correlated to the peptides derived from G3P, ENOB, and KCRM in both muscles during 14 days of storage. The peptides produced from MYG, ENOB, HBA, PGK1, and TPIS were strongly associated with improved tenderness, while those derived from major myofibrillar proteins, such as MYH1, MYH2, ACTS, and DESM, were associated with changes in tenderloin color. These results improve our understanding of the association between peptides and changes in meat quality during cold storage, indicating that proteolysis-induced peptides can be indicators of the quality characteristics of fresh and aged meat. [ABSTRACT FROM AUTHOR]

Highlights • The nutritive value of selected edible cricket powders was evaluated. • Cricket powders were found to be a good source of protein, fat, minerals and fibre. • The content of Ca, K, Mg, Na, Cu, Fe, Mn, Zn was determined. • Unique peptide markers of crickets were identified. Abstract There are various indicators, including FAO and EU sources, that edible insects could become one of the solutions to the problem of global food supply. This report was aimed at improving the knowledge on powdered crickets (Acheta domesticus). The analyses of the basic nutritional composition revealed that cricket powders were rich in protein (42.0–45.8% of dry matter) and fat (23.6–29.1% of dry matter). In terms of mineral content, CPs were rich in Ca, Mg and Fe. Most of all, the levels of Cu, Mn and Zn were especially high (2.33–4.51, 4.1–12.5, 12.8–21.8 mg/100 g of dry matter, respectively). Furthermore, the analyses into the proteins indicated that the cricket powders were treated with high temperatures and allowed the determination of four cricket-specific peptides that showed sufficient thermostability to serve as markers for authentication. [ABSTRACT FROM AUTHOR]

Highlights • Peptide markers from ribosomal proteins are diagnostic for lactic acid bacteria (LAB) • Reliable identification of lactic acid bacteria is performed by HR mass spectrometry. • LAB profiling by MS/MS is performed at genus, species and sub-species level. • The developed methodology allows the detection of LAB also in complex food matrices. Abstract Probiotic lactic acid bacteria (LAB) are generally employed in food industry because they contribute to nutritional value of fermented foods. Although knowledge of LAB composition is of high relevance for various industrial and biotechnological applications, the comprehensive identification of LAB species is sometimes technically challenging. Recently, MALDI-TOF MS-based methodologies for bacteria detection/identification in clinical diagnostics and agri-food proved to be an attractive strategy, complementary to traditional techniques for their sensitivity and specificity. In this study, we propose, for the first time, a novel methodology based on high resolution nano-LC-ESI-MS/MS for LAB identification at genus, species and sub-species level by using the sequence regions 33–52 and 72–82 of the S16 ribosomal protein as proteotypic peptide markers. The developed methodology was then applied to the analyses of buffalo and bovine whey starter cultures, thus assessing the applicability of the approach for the detection of LAB also in complex matrices. [ABSTRACT FROM AUTHOR]

In recent years, mass spectrometry (MS) has been establishing its role in the development of analytical methods for multiple allergen detection, but most analyses are being carried out on low-resolution mass spectrometers such as triple quadrupole or ion traps. In this investigation, performance provided by a high resolution (HR) hybrid quadrupole-Orbitrap™ MS platform for the multiple allergens detection in processed food matrix is presented. In particular, three different acquisition modes were compared: full-MS, targeted-selected ion monitoring with data-dependent fragmentation (t-SIM/dd2), and parallel reaction monitoring. In order to challenge the HR-MS platform, the sample preparation was kept as simple as possible, limited to a 30-min ultrasound-aided protein extraction followed by clean-up with disposable size exclusion cartridges. Selected peptide markers tracing for five allergenic ingredients namely skim milk, whole egg, soy flour, ground hazelnut, and ground peanut were monitored in home-made cookies chosen as model processed matrix. Timed t-SIM/dd2 was found the best choice as a good compromise between sensitivity and accuracy, accomplishing the detection of 17 peptides originating from the five allergens in the same run. The optimized method was validated in-house through the evaluation of matrix and processing effects, recoveries, and precision. The selected quantitative markers for each allergenic ingredient provided quantification of 60-100 μgingred/g allergenic ingredient/matrix in incurred cookies. [ABSTRACT FROM AUTHOR]

• Digestion products of shrimp-TM yielded many peptides matched T/B cell epitopes. • Digestion products of shrimp-TM in SGF profile showed a significant difference from others. • Peptides of shrimp-TM matched more T/B cell epitopes than those of fish-TM and clam-TM. • Core regions of shrimp-TM matched T/B cell epitopes are distributed along the entire chain. • A differentiating peptide of shrimp-TM (72–88) could be as a potential peptide marker. Tropomyosin (TM) is a major shellfish allergen and a minor fish allergen. Different digestion profiles affect potential allergen anaphylaxis of protein. In this study, released peptides of fish-TM, shrimp-TM, and clam-TM by in vitro digestion of simulated gastric fluid (SGF), simulated intestinal fluid (SIF), and gastrointestinal (GI) were analyzed using sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) based proteomics. Results showed that digestion products of shrimp-TM yielded a lot of peptides matched T/B cell epitopes while core regions matched epitopes were distributed along the entire chain. Pepsin or trypsin-based digestion products of shrimp-TM presented many more peptides matched T/B cell epitopes compared with those of fish-TM and clam-TM. Besides, a differentiating peptide of VEKDKALSNAEGEVAAL (72–88) overlapped T/B cell epitopes could be used as a candidate peptide marker to identify tropomyosin allergen. These findings would supply new insight into the different allergenicity of tropomyosin. [ABSTRACT FROM AUTHOR]

• High-throughput peptidomics was used to identify marker peptides in milk. • Reconstituted milk and UHT milk showed different peptide profiles. • β-Casein was more prone to protease hydrolysis compared to α s1 -casein. • Heat load and storage time affected the peptides. • The peptides can be used to monitor quality during processing and storage. An untargeted peptide profiling based on ultra-performance liquid chromatography quadrupole time-of flight mass spectrometry with chemometrics was performed to differentiate ultra-high temperature processed milk and reconstituted milk. Thirty-three marker peptides were identified, primarily released from the C- or N -terminal of β-casein and α s1 -casein. These peptides were produced by heating and protease hydrolysis. Additional heating and storage time experiments showed that the level of 18 marker peptides increased with heat load and storage time, whereas 15 peptides were solely influenced by heat load. The peptides from β-casein showed higher sensitivity to thermal stress compared to those from α s1 -casein. Additionally, eight modified peptides of casein were identified as indicators of milk thermal processing. The identified marker peptides can distinguish ultra-high temperature processed milk and reconstituted milk, and are suitable for monitoring heating processes and storage of milk. [ABSTRACT FROM AUTHOR]

The purpose of this study was to identify porcine-specific peptide markers from thermally processed meat that could differentiate pork from beef, chevon and chicken meat. In the initial stage, markers from tryptic digested protein of chilled, boiled and autoclaved pork were identified using LC–QTOF-MS. An MRM method was then established for verification. A thorough investigation of LC–QTOF-MS data showed that only seven porcine-specific peptides were consistently detected. Among these peptides, two were derived from lactate dehydrogenase, one from creatine kinase, and four from serum albumin protein. However, MRM could only detect four peptides (EVTEFAK, LVVITAGAR, FVIER and TVLGNFAAFVQK) that were consistently present in pork samples. In conclusion, meat species determination through a tandem mass spectrometry platform shows high potential in providing scientifically valid and reliable results even at peptide level. Besides, the specificity and selectivity offered by the proteomics approach also provide a robust platform for Halal authentication. [ABSTRACT FROM AUTHOR]

Abstract: In this work, we demonstrate that detection of a specific peptide marker by immunoaffinity capillary electrophoresis–mass spectrometry (IA-CE–MS) could be used to confirm the presence of recombinant human erythropoietin (rhEPO) in solution. Besides the carbohydrate content, the amino acid sequence of novel erythropoiesis stimulating protein (NESP) differs from human erythropoietin (hEPO) at five positions (Ala30Asn, His32Thr, Pro87Val, Trp88Asn, and Pro90Thr). After digesting both glycoproteins in solution by trypsin and PNGase F, two specific proteotypic peptides, EPO (77–97) and NESP (77–97) which differ in three amino acids, were selected as rhEPO and NESP markers, respectively. Both digests and their mixtures were analyzed by IA-CE–MS. The IA stationary phase was prepared from a custom made polyclonal anti-EPO (81–95) antibody immobilized on a solid support of CNBr-Sepharose 4B and was packed in a microcartridge near the inlet of the separation capillary. As the antibody was directed to a synthetic peptide EPO (81–95), only the proteotypic peptide EPO (77–97) was retained. The retained peptide was eluted, separated by electrophoresis and detected by MS. The method was specific to confirm the presence of rhEPO in solution. Although the limits of detection for the peptide marker were similar to those obtained with CE–MS (a few mg/L), these results show the potential of this novel approach to detect in the future rhEPO and its analogues selectively and unambiguously at the levels expected in biological fluids. [Copyright &y& Elsevier]

Genetic variants of human plasma alpha-1 acid glycoprotein (AGP) have been studied in cancer, compared with a group of healthy control. AGP has four genetic variants: AGP F1, F2, and S variants correspond to the ORM1 gene whereas AGP A corresponds to the ORM2 gene. The proportion of ORM1 and ORM2 variants were studied in plasma using a novel UPLC–MS method. Plasma total AGP level was 0.5 ± 0.2 g L−1 and the proportions of the ORM1 and ORM2 variants were 76.3 ± 8.2% and 23.7 ± 8.2%, respectively. In cancer plasma AGP levels increased fourfold and the proportion of ORM1 variants increased to 88.7 ± 6.8%. Changes in the proportion of genetic variants due to cancer were clearly significant, as shown by statistical analysis. Three different cancer types have been studied, lymphoma, melanoma, and ovarian cancer. The results did not show any difference depending on cancer type. The results indicate that, in accordance with prior expectations, the ORM1 variant is predominantly responsible for the acute-phase property of AGP. [ABSTRACT FROM AUTHOR]

• MS-based proteomics approaches aided in the discovery of peptide biomarkers of • A systematic screening of peptide biomarkers can improve the identification of specific-species peptide markers. • The selection of peptides is crucial as food products have high degree of variations in peptide digestion. • Porcine-specific peptide markers were identified 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. [ABSTRACT FROM AUTHOR]

• LC-QTOF-MS/MS identification of species-specific peptides was carried out. • Heat-stable peptide markers specific to rabbit were detected and identified. • 49 markers originating from 23 myofibrillar and sarcoplasmic proteins are reported. • 11 pâté-type products were tested for their compliance with labelling. Rabbit is a healthy meat, with low allergenicity and excellent nutritional properties. The global popularity of rabbit meat makes it a target for food fraud. We present a LC-QTOF-MS/MS approach for detecting and identifying rabbit-specific peptide-markers from thermally processed meat products to differentiate rabbit from other commonly-consumed animal species. We identified 49 heat-stable specific peptides. We selected the most stable markers for testing complex meat matrices by analysing pâtés-type products with a rabbit meat content ranging from 5% to 85%. Of the 49 heat-stable peptides detected in pure cooked rabbit meat, three were consistently detected in all investigated pâté samples i.e., SSVFVADPK, AFFGHYLYEVAR and PHSHPALTPEQK. Monitoring meat species other than rabbit in the examined pâtés using pork-, lamb- and chicken-specific peptides identified the presence of undeclared chicken in two samples. The results confirm that LC-QTOF-MS/MS is a suitable tool for multi-species detection in processed meat products, particularly for authentication purposes. [ABSTRACT FROM AUTHOR]

Ricin is a type II ribosome-inactivating protein toxin consisting of A and B chains linked by one interchain disulfide bond. Because of its high toxicity depending on both chains together, confirming the presence of both A and B chains of intact ricin is required during the investigation of the illegal production and application. Here, we report a novel and sensitive acetonitrile (ACN)-assisted trypsin digestion method for unambiguous identification of intact ricin by simultaneous detection of its marker peptides from A and B chains. Marker peptides were generated with a simple procedure by direct cleaving the native ricin at 45 °C for 4 h using Promega modified sequencing grade trypsin under the assistance of 10% ACN, and then directly analyzed by ultrahigh performance liquid chromatography tandem mass spectrometry. The type of trypsin was found to be one critical factor for cleavage of intact ricin based on a significant difference in the yields of specific peptides generated while using various types of trypsin. A low content of ACN in enzymatic buffer significantly reduced the digestion time from overnight to 4 h. There was commonly a better MS response of marker peptides when using the developed ACN-assisted trypsin digestion method than methanol-assisted trypsin digestion within the same 4 h. Totally, seven specific peptides with high sensitivity and specificity including three in the A-chain (TA7, TA11, and TA10) and four in the B-chain (TB6, TB14-ss-TB16, TB20, and TB18) were obtained as good marker peptides for unambiguous identification of intact ricin. The lowest concentration of native ricin for unambiguous identification was 20 ng/mL, in which three marker peptides from both the A-chain and B-chain could be measured with a minimum of three ion transitions. Combined with affinity enrichment, the developed approach was successfully applied for the measurement of intact ricin from the complicated matrix samples of the second, third, and fourth biotoxin exercises organized by the Organisation for the Prohibition of Chemical Weapons (OPCW). This study has provided a recommended detection method combined with one novel ACN-assisted trypsin digestion with MS for forensic unambiguous confirmation of trace ricin intact with high confidence.

Clostridioides difficile infection (CDI) has become the main cause of nosocomial infective diarrhoea. To survey and control the spread of different C. difficile strains, there is a need for suitable rapid tests. The aim of this study was to identify peptide/protein markers for the rapid recognition of C. difficile strains by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We analysed 44 well-characterized strains, belonging to eight different multi-locus sequence types (MLST), using ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS. The amino acid sequence of two peptide markers specific for MLST-1 and MLST-11 strains was elucidated by MALDI-TOF-MS/MS. The investigation of 2689 C. difficile genomes allowed the determination of the sensitivity and specificity of these markers. C18-solid-phased extraction was used to enrich the MLST-1 marker. Two peptide markers (m/z 4927.81 and m/z 5001.84) were identified and characterized for MLST-1 and MLST-11 strains, respectively. The MLST-1 marker was found in 786 genomes of which three did not belong to MLST-1. The MLST-11 marker was found in 319 genomes, of which 14 did not belong to MLST-11. Importantly, all MLST-1 and MLST-11 genomes were positive for their respective marker. Furthermore, a peptide marker (m/z 5015.86) specific for MLST-15 was found in 59 genomes. We translated our findings into a fast and simple method that allowed the unambiguous identification of the MLST-1 marker on a MALDI-TOF-MS platform. MALDI-FTICR MS-based peptide profiling resulted in the identification of peptide markers for C. difficile MLST-1 and MLST-11. [ABSTRACT FROM AUTHOR]

The aim of this review is to provide a brief summary of the recent applications of peptide molecules in the food area. Since, in the last few years, there has been growing interest in food authenticity, the role of peptides in this area has assumed greater importance. A fundamental part, as discussed in the article, was played by the growth of innovative analytical techniques, based on mass spectrometry (MS), which allows identifying and quantifying protein fragments in complex mixtures of different components. These procedures have been successfully applied for the identification of peptide markers in food authenticity. Because food‐derived proteins have high variability in chemical and physical properties, it is obvious that an exhaustive proteomic analysis cannot be applied by using a single technique for all objectives. The report shows some examples of various methodologies applied to different food matrices and some trivial aspects are also critically described. [ABSTRACT FROM AUTHOR]