Dissecting Ubiquitylation and DNA Damage Response Pathways in the Yeast Saccharomyces cerevisiae Using a Proteome-Wide Approach.

In response to genotoxic stress, cells evolved with a complex signaling network referred to as the DNA damage response (DDR). It is now well established that the DDR depends upon various posttranslational modifications; among them, ubiquitylation plays a key regulatory role. Here, we profiled ubiquitylation in response to the DNA alkylating agent methyl methanesulfonate (MMS) in the budding yeast Saccharomyces cerevisiae using quantitative proteomics. To discover new proteins ubiquitylated upon DNA replication stress, we used stable isotope labeling by amino acids in cell culture, followed by an enrichment of ubiquitylated peptides and LC-MS/MS. In total, we identified 1853 ubiquitylated proteins, including 473 proteins that appeared upregulated more than 2-fold in response to MMS treatment. This enabled us to localize 519 ubiquitylation sites potentially regulated upon MMS in 435 proteins. We demonstrated that the overexpression of some of these proteins renders the cells sensitive to MMS. We also assayed the abundance change upon MMS treatment of a selection of yeast nuclear proteins. Several of them were differentially regulated upon MMS treatment. These findings corroborate the important role of ubiquitin-proteasome-mediated degradation in regulating the DDR.

Caffeine Decreases Topiramate Levels in Zebrafish Larvae in a Pentylenetetrazol-Induced Seizure Model.

Epilepsy ranks as the second-most prevalent neurological disease, and is characterized by seizures resulting in neurobiological and behavioral impairment. Naturally occurring in coffee beans or tea leaves, the alkaloid caffeine (CAF) is the most prevalent global stimulant. Caffeine has been observed to influence epileptic seizures and the efficacy of antiepileptic medications, with a notable impact on topiramate (TPM). This study aimed to explore the influence of CAF on TPM's anticonvulsant effects in zebrafish larvae within a PTZ-induced seizure model, concurrently determining TPM concentrations through a sophisticated analytical approach based on ultrahigh-performance liquid chromatography and subsequent mass spectrometric detection. Zebrafish larvae four days post-fertilization were incubated for 18 h with varying doses of TPM or combinations of CAF + TPM, and locomotor activity was then assessed. Seizures were induced by introducing a PTZ solution to achieve a final concentration of 20 mM. Utilizing liquid chromatography-mass spectrometry (LC-MS/MS), TPM levels in the larvae were quantified. CAF co-administration (especially in higher doses) with TPM caused a decrease in the average locomotor activity in the larvae compared to TPM alone. Moreover, CAF decreased TPM levels in the larvae at all investigated doses. In conclusion, these findings offer a novel perspective on the interplay between CAF and TPM, shedding light on previously unexplored facets. The potential impact of CAF consumption in assisting with epileptic seizure control, unless proven otherwise, suggests a noteworthy consideration for future research and clinical practices.

Novel multi-target ligands of dopamine and serotonin receptors for the treatment of schizophrenia based on indazole and piperazine scaffolds-synthesis, biological activity, and structural evaluation.

Schizophrenia is a chronic mental disorder that is not satisfactorily treated with available antipsychotics. The presented study focuses on the search for new antipsychotics by optimising the compound D2AAK3, a multi-target ligand of G-protein-coupled receptors (GPCRs), in particular D2, 5-HT1A, and 5-HT2A receptors. Such receptor profile may be beneficial for the treatment of schizophrenia. Compounds 1-16 were designed, synthesised, and subjected to further evaluation. Their affinities for the above-mentioned receptors were assessed in radioligand binding assays and efficacy towards them in functional assays. Compounds 1 and 10, selected based on their receptor profile, were subjected to in vivo tests to evaluate their antipsychotic activity, and effect on memory and anxiety processes. Molecular modelling was performed to investigate the interactions of the studied compounds with D2, 5-HT1A, and 5-HT2A receptors on the molecular level. Finally, X-ray study was conducted for compound 1, which revealed its stable conformation in the solid state.

Optimization of the Zebrafish Larvae Pentylenetetrazol-Induced Seizure Model for the Study of Caffeine and Topiramate Interactions.

Epilepsy is a common neurological disorder characterized by seizures that cause neurobiological and behavioral impairment. Caffeine (CAF), which is the most widely consumed stimulant in the world, is reported to influence epileptic seizures and antiepileptic drugs, especially topiramate (TPM). The aim of the study was to optimize the zebrafish larvae pentylenetetrazol-induced seizure model for the study of CAF and TPM interactions, which include the determination of dose space, and the delivery of an analytical method for monitoring CAF, TPM, and CAF metabolite paraxanthine (PAR) in Zebrafish larvae. Methods: The zebrafish larvae, 4 days post-fertilization, were incubated for 18 h with CAF, TPM, or CAF + TPM, with subsequent locomotor activity assessment. Seizures were evoked by adding PTZ solution to obtain a final concentration of 20 mM. Subsequently, the liquid chromatography-mass spectrometry (LC-MS/MS) analytical method was used to simultaneously assess the levels of both CAF and TPM in the larvae. CAF (50 mg/L) and TPM (75 µM) given separately decreased the average larvae locomotor activity compared to the PTZ group but, however, were not able to lower it to the control level. Co-administration of 25 mg/L CAF and 50 µM TPM suppressed the activity to the same level. Adding 25 µM TPM to 50 mg/L CAF decrease the measured CAF level in the larvae. Until proven otherwise, CAF consumption should be regarded as a potential determinant in the modulation of TPM's efficacy in the management of epileptic seizures. The optimized model will contribute to the standardization of studying CAF and TPM interactions and building the understanding of the molecular bases of the interaction.

LC-MS Metabolomic Profiling of Five Types of Unrefined, Cold-Pressed Seed Oils to Identify Markers to Determine Oil Authenticity and to Test for Oil Adulteration.

The authenticity of food products marketed as health-promoting foods-especially unrefined, cold-pressed seed oils-should be controlled to ensure their quality and safeguard consumers and patients. Metabolomic profiling using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF) was employed to identify authenticity markers for five types of unrefined, cold-pressed seed oils: black seed oil (Nigella sativa L.), pumpkin seed oil (Cucurbita pepo L.), evening primrose oil (Oenothera biennis L.), hemp oil (Cannabis sativa L.) and milk thistle oil (Silybum marianum). Of the 36 oil-specific markers detected, 10 were established for black seed oil, 8 for evening primrose seed oil, 7 for hemp seed oil, 4 for milk thistle seed oil and 7 for pumpkin seed oil. In addition, the influence of matrix variability on the oil-specific metabolic markers was examined by studying binary oil mixtures containing varying volume percentages of each tested oil and each of three potential adulterants: sunflower, rapeseed and sesame oil. The presence of oil-specific markers was confirmed in 7 commercial oil mix products. The identified 36 oil-specific metabolic markers proved useful for confirming the authenticity of the five target seed oils. The ability to detect adulterations of these oils with sunflower, rapeseed and sesame oil was demonstrated.

Comparison of the Determination of Some Antihypertensive Drugs in Clinical Human Plasma Samples by Solvent Front Position Extraction and Precipitation Modes.

The determination of the selected antihypertensive drugs in human plasma samples with the novel solvent front position extraction (SFPE) technique is presented. The SFPE procedure combined with LC-MS/MS analysis was used for the first time to prepare a clinical sample containing the drugs mentioned above from different therapeutic groups. The effectiveness of our approach was compared with the precipitation method. The latter technique is usually used to prepare biological samples in routine laboratories. During the experiments, the substances of interest and the internal standard were separated from other matrix components using a prototype horizontal chamber for thin-layer chromatography/high-performance thin-layer chromatography (TLC/HPTLC) with a moving pipette powered by a 3D mechanism, which distributed the solvent on the adsorbent layer. Detection of the six antihypertensive drugs was performed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode. Results obtained by SFPE were very satisfactory (linearity R2 ≥ 0.981; %RSD ≤ 6%; LOD and LOQ were in the range of 0.06-9.78 ng/mL and 0.17-29.64 ng/mL, respectively). The recovery was in the range of 79.88-120.36%. Intra-day and inter-day precision had a percentage coefficient of variation (CV) in the range of 1.10-9.74%. The procedure is simple and highly effective. It includes the automation of TLC chromatogram development, which significantly reduced the number of manual operations performed, the time of sample preparation and solvent consumption.

Development and Characterization of Novel Selective, Non-Basic Dopamine D2 Receptor Antagonists for the Treatment of Schizophrenia.

The dopamine D2 receptor, which belongs to the family of G protein-coupled receptors (GPCR), is an important and well-validated drug target in the field of medicinal chemistry due to its wide distribution, particularly in the central nervous system, and involvement in the pathomechanism of many disorders thereof. Schizophrenia is one of the most frequent diseases associated with disorders in dopaminergic neurotransmission, and in which the D2 receptor is the main target for the drugs used. In this work, we aimed at discovering new selective D2 receptor antagonists with potential antipsychotic activity. Twenty-three compounds were synthesized, based on the scaffold represented by the D2AAK2 compound, which was discovered by our group. This compound is an interesting example of a D2 receptor ligand because of its non-classical binding to this target. Radioligand binding assays and SAR analysis indicated structural modifications of D2AAK2 that are possible to maintain its activity. These findings were further rationalized using molecular modeling. Three active derivatives were identified as D2 receptor antagonists in cAMP signaling assays, and the selected most active compound 17 was subjected to X-ray studies to investigate its stable conformation in the solid state. Finally, effects of 17 assessed in animal models confirmed its antipsychotic activity in vivo.

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 ß-enolase are the main protein sources of heat-stable markers. © 2019 John Wiley & Sons Ltd. Mass Spec Rev.

Reactions of 3-Hydroxy-2-phenyl-1H-benzo[e]isoindol-1-one: A Route to 3-Hydroxy-/3-anilinobenzo[e]indan-1-ones and Benzo[f]phthalazin-1(2H)-ones.

New hydroxy- and anilinoindanone derivatives 3 and 4 were synthesized starting from 3-hydroxybenzo[e]isoindolinone 1 via the addition of alkyllithium (s-BuLi, n-BuLi, MeLi or i-PrLi) to the carbonyl group, followed by lactam ring opening and, finally, an intramolecular cyclization leading to target compounds. The same starting material was used for the preparation of the new benzo[f]phthalazinone derivatives 12-16 through multi-step reactions. The target derivative 16 was obtained from the corresponding bromolactam 15 by the Buchwald-Hartwig amination. Structures of the obtained compounds were confirmed by the NMR spectra.

Shotgun Lipidomic Analysis for Differentiation of Niche Cold Pressed Oils.

The fast-growing food industry is bringing significant number of new products to the market. To protect consumers' health and rights, it is crucial that food control laboratories are able to ensure reliable quality testing, including product authentication and detection of adulterations. In our study, we applied a fast and eco-friendly method based on shotgun-lipidomic mass spectrometry for the authentication of niche edible oils. Comprehensive lipid profiles of camelina (CA), flax (FL) and hemp (HP) seed oils were obtained. With the aid of principal component analysis (PCA), it was possible to detect and distinguish each of them based on their lipid profiles. Lipidomic markers characteristic ofthe oils were also identified, which can be used as targets and expedite development of new multiplexed testing methods.

Influence of Complexation of Thiosemicarbazone Derivatives with Cu (II) Ions on Their Antitumor Activity against Melanoma Cells.

A series of thiosemicarbazone derivatives was prepared and their anti-tumor activity in vitro was tested. The X-ray investigation performed for compounds T2, T3 and T5 confirmed the synthesis pathway and assumed molecular structures of analyzed thiosemicarbazones. The conformational preferences of the thiosemicarbazone system were characterized using theoretical calculations by AM1 method. Selected compounds were converted into complexes of Cu (II) ions. The effect of complexing on anti-tumor activity has been investigated. The copper(II) complexes, with Schiff bases T1, T10, T12, T13, and T16 have been synthesized and characterized by chemical and elemental analysis, FTIR spectroscopy and TGA method. Thermal properties of coordination compounds were studied using TG-DTG techniques under dry air atmosphere. G361, A375, and SK-MEL-28 human melanoma cells and BJ human normal fibroblast cells were treated with tested compounds and their cytotoxicity was evaluated with MTT test. The compounds with the most promising anti-tumour activity were then selected and their cytotoxicity was verified with cell cycle analysis and apoptosis/necrosis detection. Additionally, DNA damages in the form of a basic sites presence and the expression of oxidative stress and DNA damage response genes were evaluated. The obtained results indicate that complexation of thiosemicarbazone derivatives with Cu (II) ions improves their antitumor activity against melanoma cells. The observed cytotoxic effect is associated with DNA damage and G2/M phase of cell cycle arrest as well as disorders of the antioxidant enzymes expression.

Effects of Physical and Chemical Factors on the Structure of Gluten, Gliadins and Glutenins as Studied with Spectroscopic Methods.

This review presents applications of spectroscopic methods, infrared and Raman spectroscopies in the studies of the structure of gluten network and gluten proteins (gliadins and glutenins). Both methods provide complimentary information on the secondary and tertiary structure of the proteins including analysis of amide I and III bands, conformation of disulphide bridges, behaviour of tyrosine and tryptophan residues, and water populations. Changes in the gluten structure can be studied as an effect of dough mixing in different conditions (e.g., hydration level, temperature), dough freezing and frozen storage as well as addition of different compounds to the dough (e.g., dough improvers, dietary fibre preparations, polysaccharides and polyphenols). Additionally, effect of above mentioned factors can be determined in a common wheat dough, model dough (prepared from reconstituted flour containing only wheat starch and wheat gluten), gluten dough (lack of starch), and in gliadins and glutenins. The samples were studied in the hydrated state, in the form of powder, film or in solution. Analysis of the studies presented in this review indicates that an adequate amount of water is a critical factor affecting gluten structure.

Comprehensive Thermal Characteristics of Different Cultivars of Flaxseed Oil (Linum usittatissimum L.).

The aim of this study was to describe the thermal properties of selected cultivars of flaxseed oil by the use of the differential scanning calorimetry (DSC) technique. The crystallization and melting profiles were analyzed depending on different scanning rates (1, 2, 5 °C/min) as well as oxidative induction time (OIT) isothermally at 120 °C and 140 °C, and oxidation onset temperatures (Ton) at 2 and 5 °C/min were measured. The crystallization was manifested as a single peak, differing for a cooling rate of 1 and 2 °C/min. The melting curves were more complex with differences among the cultivars for a heating rate of 1 and 2 °C/min, while for 5 °C/min, the profiles did not differ, which could be utilized in analytics for profiling in order to assess the authenticity of the flaxseed oil. Moreover, it was observed that flaxseed oil was highly susceptible to thermal oxidation, and its stability decreased with increasing temperature and decreasing heating rate. Significant negative linear correlations were found between unsaturated fatty acid content (C18:2, C18:3 n-3) and DSC parameters (OIT, Ton). Principal component analysis (PCA) also established a strong correlation between total oxidation value (TOTOX), peroxide value (PV) and all DSC parameters of thermo-oxidative stability.

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.

Comprehensive Insight into Chemical Stability of Important Antidiabetic Drug Vildagliptin Using Chromatography (LC-UV and UHPLC-DAD-MS) and Spectroscopy (Mid-IR and NIR with PCA).

During forced degradation, the intrinsic stability of active pharmaceutical ingredients (APIs) could be determined and possible impurities that would occur during the shelf life of the drug substance or the drug product could be estimated. Vildagliptin belongs to relatively new oral antidiabetic drugs named gliptins, inhibiting dipeptidyl peptidase 4 (DPP-4) and prolonging the activities of the endogenous incretin hormones. At the same time, some gliptins were shown as prone to degradation under specific pH and temperature conditions, as well as in the presence of some reactive excipients. Thus, forced degradation of vildagliptin was performed at high temperature in extreme pH and oxidative conditions. Then, selective LC-UV was used for quantitative determination of non-degraded vildagliptin in the presence of its degradation products and for degradation kinetics. Finally, identification of degradation products of vildagliptin was performed using an UHPLC-DAD-MS with positive ESI. Stability of vildagliptin was also examined in the presence of pharmaceutical excipients, using mid-IR and NIR with principal component analysis (PCA). At 70 °C almost complete disintegration of vildagliptin occurred in acidic, basic, and oxidative media. What is more, high degradation of vildagliptin following the pseudo first-order kinetics was observed at room temperature with calculated k values 4.76 × 10-4 s-1, 3.11 × 10-4 s-1, and 1.73 × 10-4 s-1 for oxidative, basic and acidic conditions, respectively. Next, new degradation products of vildagliptin were detected using UHPLC-DAD-MS and their molecular structures were proposed. Three degradants were formed under basic and acidic conditions, and were identified as [(3-hydroxytricyclo- [3.3.1.13,7]decan-1-yl)amino]acetic acid, 1-{[(3-hydroxytricyclo[3.3.1.13,7]decan-1-yl)amino]acetyl}-pyrrolidine-2-carboxylic acid and its O-methyl ester. The fourth degradant was formed in basic, acidic, and oxidative conditions, and was identified as 1-{[(3-hydroxytricyclo[3.3.1.13,7]-decan-1-yl)amino]acetyl}pyrrolidine-2-carboxamide. When stability of vildagliptin was examined in the presence of four excipients under high temperature and humidity, a visible impact of lactose, mannitol, magnesium stearate, and polyvinylpirrolidone was observed, affecting-NH- and CO groups of the drug. The obtained results (kinetic parameters, interactions with excipients) may serve pharmaceutical industry to prevent chemical changes in final pharmaceutical products containing vildagliptin. Other results (e.g., identification of new degradation products) may serve as a starting point for qualifying new degradants of vildagliptin as it is related to substances in pharmacopoeias.

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

BACKGROUND: 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. RESULTS: 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). CONCLUSIONS: 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.

Amino- and polyaminophthalazin-1(2H)-ones: synthesis, coordination properties, and biological activity.

Amino- and polyaminophthalazinones were synthesized by the palladium-catalyzed amination (alkyl- and arylamines, polyamines) of 4-bromophthalazinones in good yields. The coordinating properties of selected aminophthalazinones towards Cu(II) ions were investigated and the participation of the nitrogen atoms in the complexation of the metal ion was shown. A biological screening of the potential cytotoxicity of selected synthesized compounds on HT-29 and PC-3 cell lines, as well as on the L-929 cell line, proved that some amino derivatives of phthalazinone show interesting anticancer activities. The detailed synthesis, spectroscopic data, and biological assays are reported.

1,2,4-Triazolin-5-thione derivatives with anticancer activity as CK1γ kinase inhibitors.

The optimization and synthesis of new CK2 and CK1 inhibitors are the basis for the development of new therapeutic strategies for the treatment of cancer and neurodegenerative disorders associated with overexpression and abnormal functioning of these enzymes. Triazole derivatives appear to be especially interesting as potential kinase inhibitors. In this context we synthesized a series of 1,2,4-triazolin-5-thione derivatives as CK1γ kinase inhibitors. The antiproliferative activity of synthesized compounds was assessed against cancer cells: human lung adenocarcinoma (A549), human hepatoma (HepG2), and human breast adenocarcinoma (MCF-7). Compound 1 exhibited antiproliferative potency against A549 cancer cells and was characterized by a selective antiproliferative effect. Additionally, this compound has high apoptotic activity against A549, HepG2, MCF-7 cells and induced only slight amount of necrotic cells in these cell lines. In order to decipher the mechanism of anticancer activity of the studied compounds PASS software was used and these compounds were assayed for the inhibition of CK1γ and CK2α kinases. The reported series of 1,2,4-triazolin-5-thiones inhibits CK1γ and CK2α kinases in micromolar range. The most active compound shows activity against isoform γ3 which at concentration of 50 µM reduced the kinase activity by 69% while at 100 µM by 80%. CK2α was found to be less susceptible to the effects of the triazoles tested, as the reduction in kinase activity by 29% was observed for compound 15, and by 27% for compound 1 only at the concentration of 100 µM. The inhibition of CK1γ and CK2α kinases was rationalized using molecular docking.

A Mass Spectrometry-Based Approach for Characterization of Red, Blue, and Purple Natural Dyes.

Effective analytical approaches for the identification of natural dyes in historical textiles are mainly based on high-performance liquid chromatography coupled with spectrophotometric detection and tandem mass spectrometric detection with electrospray ionization (HPLC-UV-Vis-ESI MS/MS). Due to the wide variety of dyes, the developed method should include an adequate number of reference color compounds, but not all of them are commercially available. Thus, the present study was focused on extending of the universal analytical HPLC-UV-Vis-ESI MS/MS approach to commercially unavailable markers of red, purple, and blue dyes. In the present study, HPLC-UV-Vis-ESI MS/MS was used to characterize the colorants in ten natural dyes (American cochineal, brazilwood, indigo, kermes, lac dye, logwood, madder, orchil, Polish cochineal, and sandalwood) and, hence, to extend the analytical method for the identification of natural dyes used in historical objects to new compounds. Dye markers were identified mostly on the basis of triple quadrupole MS/MS spectra. In consequence, the HPLC-UV-Vis-ESI MS/MS method with dynamic multiple reaction monitoring (dMRM) was extended to the next 49 commercially unavailable colorants (anthraquinones and flavonoids) in negative ion mode and to 11 (indigoids and orceins) in positive ion mode. These include protosappanin B, protosappanin E, erythrolaccin, deoxyerythrolaccin, nordamnacanthal, lucidin, santalin A, santalin B, santarubin A, and many others. Moreover, high-resolution QToF MS data led to the establishment of the complex fragmentation pathways of α-, ß-, and γ- aminoorceins, hydroxyorceins, and aminoorceinimines extracted from wool dyed with Roccella tinctoria DC. The developed approach has been tested in the identification of natural dyes used in 223 red, purple, and blue fibers from 15th- to 17th-century silk textiles. These European and Near Eastern textiles have been used in vestments from the collections of twenty Krakow churches.

LC⁻Q⁻TOF⁻MS/MS Identification of Specific Non-Meat Proteins and Peptides in Beef Burgers.

Beef burgers are a popular food choice, due to their taste and convenience. The extensive range of beef burgers with different flavours currently offered on the market is adding to their growing consumption. This study detected and identified specific non-meat proteins and peptide markers originating from functional preparations, i.e., powdered mixes of protein additives and spices, used as meat substitutes in the production of ready-to-cook beef burgers. Twenty-eight soy proteins, including isoforms (nine milk-, three pea- and one beetroot-specific protein) were found concurrently with a set of peptide markers unique to soy glycinin and ß-conglycinin, pea vicilin and provicilin, milk αS1-casein, ß-lactoglobulin, as well as beetroot elongation factor 2. Soy and beetroot proteins and peptides were observed in all burgers containing additives. Milk and pea proteins were included in powdered mixes but were not detected in burgers, indicating that their content was below the limit of detection. The study demonstrates that the proposed method can be implemented to analyse protein additives in cooked burgers; however, the presence of low amounts of additives, below 1⁻2%, should be further confirmed by using a more sensitive triple quadrupole instrument.