Matrix Selection Strategies for MALDI-TOF MS/MS Characterization of Cyclic Tetrapyrroles in Blood and Food Samples.

Cyclic tetrapyrrole derivatives such as porphyrins, chlorins, corrins (compounds with a corrin core), and phthalocyanines are a family of molecules containing four pyrrole rings usually coordinating a metal ion (Mg, Cu, Fe, Zn, etc.). Here, we report the characterization of some representative cyclic tetrapyrrole derivatives by MALDI-ToF/ToF MS analyses, including heme b and c, phthalocyanines, and protoporphyrins after proper matrix selection. Both neutral and acidic matrices were evaluated to assess potential demetallation, adduct formation, and fragmentation. While chlorophylls exhibited magnesium demetallation in acidic matrices, cyclic tetrapyrroles with Fe, Zn, Co, Cu, or Ni remained steadfast against demetallation across all conditions. Phthalocyanines and protoporphyrins were also detectable without a matrix using laser desorption ionization (LDI); however, the incorporation of matrices achieved the highest ionization yield, enhanced sensitivity, and negligible fragmentation. Three standard proteins, i.e., myoglobin, hemoglobin, and cytochrome c, were analyzed either intact or enzymatically digested, yielding heme b and heme c ions along with accompanying peptides. Furthermore, we successfully detected and characterized heme b in real samples, including blood, bovine and cod liver, and mussel. As a result, MALDI MS/MS emerged as a powerful tool for straightforward cyclic tetrapyrrole identification, even in highly complex samples. Our work paves the way for a more comprehensive understanding of cyclic tetrapyrroles in biological and industrial settings, including the geochemical field, as these compounds are a source of significant geological and geochemical information in sediments and crude oils.

Food allergen detection by mass spectrometry: From common to novel protein ingredients.

Food allergens are molecules, mainly proteins, that trigger immune responses in susceptible individuals upon consumption even when they would otherwise be harmless. Symptoms of a food allergy can range from mild to acute; this last effect is a severe and potentially life-threatening reaction. The European Union (EU) has identified 14 common food allergens, but new allergens are likely to emerge with constantly changing food habits. Mass spectrometry (MS) is a promising alternative to traditional antibody-based assays for quantifying multiple allergenic proteins in complex matrices with high sensitivity and selectivity. Here, the main allergenic proteins and the advantages and drawbacks of some MS acquisition protocols, such as multiple reaction monitoring (MRM) and data-dependent analysis (DDA) for identifying and quantifying common allergenic proteins in processed foodstuffs are summarized. Sections dedicated to novel foods like microalgae and insects as new sources of allergenic proteins are included, emphasizing the significance of establishing stable marker peptides and validated methods using database searches. The discussion involves the in-silico digestion of allergenic proteins, providing insights into their potential impact on immunogenicity. Finally, case studies focussing on microalgae highlight the value of MS as an effective analytical tool for ensuring regulatory compliance throughout the food control chain.

Development, Analytical Characterization, and Bioactivity Evaluation of Boswellia serrata Extract-Layered Double Hydroxide Hybrid Composites.

Boswellia serrata Roxb. extract (BSE), rich in boswellic acids, is well known as a potent anti-inflammatory natural drug. However, due to its limited aqueous solubility, BSE inclusion into an appropriate carrier, capable of improving its release in the biological target, would be highly desirable. Starting with this requirement, new hybrid composites based on the inclusion of BSE in a lamellar solid layered double hydroxide (LDH), i.e., magnesium aluminum carbonate, were developed and characterized in the present work. The adopted LDH exhibited a layered crystal structure, comprising positively charged hydroxide layers and interlayers composed of carbonate anions and water molecules; thus, it was expected to embed negatively charged boswellic acids. In the present case, a calcination process was also adopted on the LDH to increase organic acid loading, based on the replacement of the original inorganic anions. An accurate investigation was carried out by TGA, PXRD, FT-IR/ATR, XPS, SEM, and LC-MS to ascertain the nature, interaction, and quantification of the active molecules of the vegetal extract loaded in the developed hybrid materials. As a result, the significant disruption of the original layered structure was observed in the LDH subjected to calcination (LDHc), and this material was able to include a higher amount of organic acids when its composite with BSE was prepared. However, in vitro tests on the composites' bioactivity, expressed in terms of antimicrobial and anti-inflammatory activity, evidenced LDH-BSE as a better material compared to BSE and to LDHc-BSE, thus suggesting that, although the embedded organic acid amount was lower, they could be more available since they were not firmly bound to the clay. The composite was able to significantly decrease the number of viable pathogens such as Escherichia coli and Staphylococcus aureus, as well as the internalization of toxic active species into human cells imposing oxidative stress, in comparison to the BSE.

Methyl carbamates of phosphatidylethanolamines and phosphatidylserines reveal bacterial contamination in mitochondrial lipid extracts of mouse embryonic fibroblasts.

The occurrence of methyl carbamates of phosphatidylethanolamines and phosphatidylserines in the lipid extract of mitochondria obtained from mouse embryonic fibroblasts was ascertained by hydrophilic interaction liquid chromatography with electrospray ionization single and multi-stage mass spectrometry, performed using sinergically a high resolution (quadrupole-Orbitrap) and a low resolution (linear ion trap) spectrometer. Two possible routes to the synthesis of methyl carbamates of phospholipids were postulated and evaluated: (i) a chemical transformation involving phosgene, occurring as a photooxidation by-product in the chloroform used for lipid extraction, and methanol, also used for the latter; (ii) an enzymatic methoxycarbonylation reaction due to an accidental bacterial contamination, that was unveiled subsequently on the murine mitochondrial sample. A specific lipid extraction performed on a couple of standard phosphatidyl-ethanolamines/-serines, based on purposely photo-oxidized chloroform and deuterated methanol, indicated route (i) as negligible in the specific case, thus highlighting the enzymatic route related to bacterial contamination as the most likely source of methyl carbamates. The unambiguous recognition of the latter might represent the starting point toward a better understanding of their generation in biological systems and a minimization of their occurrence when an artefactual formation is ascertained.

Strategies for the analysis of arsenolipids in marine foods: A review.

Arsenic-containing lipids, also named arsenolipids (AsLs), are a group of organic compounds usually found in a variety of marine organisms such as fish, algae, shellfish, marine oils, and microorganisms. Numerous AsLs have been recognised so far, from simple compounds such as arsenic fatty acids (AsFAs), arsenic hydrocarbons (AsHCs), and trimethylarsenio fatty alcohols (TMAsFOHs) to more complex arsenic-containing species, of which arsenophospholipids (AsPLs) are a case in point. Mass spectrometry, both as inductively coupled plasma (ICP-MS) and liquid chromatography coupled by an electrospray source (LC-ESI-MS), was applied to organic arsenicals playing a key role in extending and refining the characterisation of arsenic-containing lipids in marine organisms. Herein, upon the introduction of a systematic notation for AsLs and a brief examination of their toxicity and biological role, the most relevant literature concerning the characterisation of AsLs in marine organisms, including edible ones, is reviewed. The use of both ICP-MS and ESI-MS coupled with reversed-phase liquid chromatography (RPLC) has brought significant advancements in the field. In the case of ESI-MS, the employment of negative polarity and tandem MS analyses has further enhanced these advancements. One notable development is the identification of the m/z 389.0 ion ([AsC10H19O9P]-) as a diagnostic product ion of AsPLs, which is obtained from the fragmentation of the deprotonated forms of AsPLs ([M - H]-). The pinpointing product ions offer the possibility of determining the identity and regiochemistry of AsPL side chains. Advanced MS-based analytical methods may contribute remarkably to the understanding of the chemical diversity characterising the metalloid As in natural organic compounds of marine organisms.

Uncovering heterogeneity of anacardic acids from pistachio shells: A novel approach for structural characterization.

Anacardic acids (AnAs) are important secondary metabolites that occur primarily in plants of the Anacardiaceae family, such as pistachio (Pistacia vera L.). Some AnAs have been associated with health benefits, and the position of the CC double bonds is a crucial feature of these metabolites. Herein, we propose a new strategy based on RPLC separation and detection by ESI-MS/MS, preceded by an epoxidation reaction. The procedure was applied to the green extracts of lignified pistachio shells, and a mixture of AnAs bearing alkyl chains 13:0, 15:0, and 17:1 emerged as prevailing. As positional isomers of AnA 15:1 (Δ8 and Δ6) and AnAs 17:1 (Δ10 and Δ8) were identified for the first time, their discovery paves the way to the systematic study of their potential health-beneficial effects. The developed method was validated and applied to quantify AnAs in pistachio ethanolic extract, showing contents higher than 10 mg/ 100 g of biomass.

PE, or not PE, that is the question: The case of overlooked lyso-N-acylphosphatidylethanolamines.

RATIONALE: Lyso derivatives of N-acyl-1,2-diacylglycero-3-phosphoethanolamines (L-NAPEs) are a lipid class mostly expressed in vegetables during stress and tissue damage that is involved in the synthesis of the lipid mediator N-acylethanolamines. L-NAPEs can be challenging to distinguish from isomeric phosphatidylethanolamines (PEs), especially in extracted complex samples where they could be confused with abundant PEs. METHODS: In this study, hydrophilic interaction liquid chromatography with electrospray ionization hyphenated with (tandem) mass spectrometry (MS) was proposed to distinguish L-NAPEs and PEs as deprotonated molecules, [M - H]─ , using both high-resolution/accuracy Fourier transform MS and low-resolution linear ion trap (LIT) mass analyzers. MS3 experiments of [M - H - KE]─ as precursor ions (KE, ketene loss) using the LIT instrument allowed us to distinguish between isomeric L-NAPE and PE species. RESULTS: Regiochemical rules were proposed working on enzymatically synthesized L-NAPEs. A few key differences in MS/MS spectra, including abnormal intensity of acyl chain losses as fatty acids, the presence of N-acylphosphoethanolamine ions, and diagnostic ions of the polar head, were disclosed. Additionally, MS3 spectra of [M - H - KE]─ as precursor ions allowed us to confirm the identification of L-NAPE species. The proposed rules were applied to samples extracted from tomato by-products including stems and leaves. CONCLUSIONS: Overall, our methodology is demonstrated as a robust approach to recognizing L-NAPEs in complex samples. L-NAPEs 18:2-N-18:2, 18:2-N-18:3, 18:3-N-18:2, and 18:2-N-18:1 were the prevailing compounds in the analyzed tomato samples, accounting for more than 90%. In summary, a reliable method for identifying L-NAPEs in complex samples is described. The proposed method could prevent overlooking L-NAPEs and overestimating isomeric PE species in future lipid analyses.

Hydrogen/Deuterium Exchange Mass Spectrometry for Probing the Isomeric Forms of Oleocanthal and Oleacin in Extra Virgin Olive Oils.

Reversed-phase liquid chromatography and electrospray ionization with Fourier-transform single and tandem mass spectrometry (RPLC-ESI-FTMS and FTMS/MS) were employed for the structural characterization of oleocanthal (OLEO) and oleacin (OLEA), two of the most important bioactive secoiridoids occurring in extra virgin olive oils (EVOOs). The existence of several isoforms of OLEO and OLEA was inferred from the chromatographic separation, accompanied, in the case of OLEA, by minor peaks due to oxidized OLEO recognized as oleocanthalic acid isoforms. The detailed analysis of the product ion tandem MS spectra of deprotonated molecules ([M-H]-) was unable to clarify the correlation between chromatographic peaks and specific OLEO/OLEA isoforms, including two types of predominant dialdehydic compounds, named Open Forms II, containing a double bond between carbon atoms C8 and C10, and a group of diasteroisomeric closed-structure (i.e., cyclic) isoforms, named Closed Forms I. This issue was addressed by H/D exchange (HDX) experiments on labile H atoms of OLEO and OLEA isoforms, performed using deuterated water as a co-solvent in the mobile phase. HDX unveiled the presence of stable di-enolic tautomers, in turn providing key evidence for the occurrence, as prevailing isoforms, of Open Forms II of OLEO and OLEA, different from those usually considered so far as the main isoforms of both secoiridoids (having a C=C bond between C8 and C9). It is expected that the new structural details inferred for the prevailing isoforms of OLEO and OLEA will help in understanding the remarkable bioactivity exhibited by the two compounds.

Multi-Target Detection of Nuts and Peanuts as Hidden Allergens in Bakery Products through Bottom-Up Proteomics and High-Resolution Mass Spectrometry.

Due to the growing global incidence of allergy to nuts and peanuts, the need for better protection of consumers sensitive to those products is constantly increasing. The best strategy to defend them against adverse immunological reactions still remains the total removal of those products from their diet. However, nuts and peanuts traces can also be hidden in other food products, especially processed ones, such as bakery products, because of cross-contamination occurring during production. Precautionary labelling is often adopted by producers to warn allergic consumers, usually without any evaluation of the actual risk, which would require a careful quantification of nuts/peanuts traces. In this paper, the development of a multi-target method based on liquid chromatography-tandem high resolution mass spectrometry (LC-MS, MS/MS), able to detect traces of five nuts species (almonds, hazelnuts, walnuts, cashews and pistachios) and of peanuts in an in-house incurred bakery product (cookie) through a single analysis is described. Specifically, allergenic proteins of the six ingredients were used as the analytical targets, and the LC-MS responses of selected peptides resulting from their tryptic digestion, after extraction from the bakery product matrix, were exploited for quantification, following a bottom-up approach typical of proteomics. As a result, nuts/peanuts could be detected/quantified down to mg·kg-1 levels in the model cookie, thus opening interesting perspectives for the quantification of hidden nuts/peanuts in bakery products and, consequently, for a more rational use of precautionary labelling.

Triacylglycerols hydrolysis and hydroxy- and epoxy-fatty acids release during lactic fermentation of plant matrices: An extensive study showing inter- and intra-species capabilities of lactic acid bacteria.

This study aims to show that lactic fermentation by selected starters can enrich plant matrices with hydroxy- and oxo-fatty acids. The behavior of 31 lactic acid bacteria strains was investigated during the fermentation of Persian walnut, which was selected as a model growth substrate due to its inherent lipids content. The content of the following free fatty acids increased in the majority of the fermented walnut samples: linoleic, α-linolenic, palmitic, and oleic acids. The increase of diacylglycerols and, especially, monoacylglycerols levels in fermented walnuts confirmed that strain-specific bacterial lipolytic activities hydrolyzed triacylglycerols during walnut fermentation. Twelve hydroxylated or epoxidized derivatives arising from oleic, linoleic, and linolenic fatty acids, in five groups of isomeric compounds, were also identified. In addition to the better-known lactobacilli, certain strains of Weissella cibaria, Leuconostoc mesenteroides, and Enterococcus faecalis emerged for their lipolytic activities and ability to release hydroxy- and epoxy-fatty acids during walnut fermentation.

All Ion Fragmentation Analysis Enhances the Untargeted Profiling of Glucosinolates in Brassica Microgreens by Liquid Chromatography and High-Resolution Mass Spectrometry.

An analytical approach based on reversed-phase liquid chromatography coupled to electrospray ionization Fourier-transform mass spectrometry in negative ion mode (RPLC-ESI-(-)-FTMS) was developed for the untargeted characterization of glucosinolates (GSL) in the polar extracts of four Brassica microgreen crops, namely, garden cress, rapeseed, kale, and broccoli raab. Specifically, the all ion fragmentation (AIF) operation mode enabled by a quadrupole-Orbitrap mass spectrometer, i.e., the systematic fragmentation of all ions generated in the electrospray source, followed by the acquisition of an FTMS spectrum, was exploited. First, the best qualifying product ions for GSL were recognized from higher-energy collisional dissociation (HCD)-FTMS2 spectra of representative standard GSL. Extracted ion chromatograms (EIC) were subsequently obtained for those ions from RPLC-ESI(-)-AIF-FTMS data referred to microgreen extracts, by plotting the intensity of their signals as a function of retention time. The alignment of peaks detected in the EIC traces was finally exploited for the recognition of peaks potentially related to GSL, with the EIC obtained for the sulfate radical anion [SO4]•- (exact m/z 95.9523) providing the highest selectivity. Each putative GSL was subsequently characterized by HCD-FTMS2 analyses and by collisionally induced dissociation (CID) multistage MSn (n = 2, 3) acquisitions based on a linear ion trap mass spectrometer. As a result, up to 27 different GSLs were identified in the four Brassica microgreens. The general method described in this work appears as a promising approach for the study of GSL, known and novel, in plant extracts.

Characterization of anti-proliferative and anti-oxidant effects of nano-sized vesicles from Brassica oleracea L. (Broccoli).

In this in vitro study, we test our hypothesis that Broccoli-derived vesicles (BDVs), combining the anti-oxidant properties of their components and the advantages of their structure, can influence the metabolic activity of different cancer cell lines. BDVs were isolated from homogenized fresh broccoli (Brassica oleracea L.) using a sucrose gradient ultracentrifugation method and were characterized in terms of physical properties, such as particle size, morphology, and surface charge by transmission electron microscopy (TEM) and laser doppler electrophoresis (LDE). Glucosinolates content was assessed by RPLC-ESI-MS analysis. Three different human cancer cell lines (colorectal adenocarcinoma Caco-2, lung adenocarcinoma NCI-H441 and neuroblastoma SHSY5Y) were evaluated for metabolic activity by the MTT assay, uptake by fluorescence and confocal microscopy, and anti-oxidant activity by a fluorimetric assay detecting intracellular reactive oxygen species (ROS). Three bands were obtained with average size measured by TEM based size distribution analysis of 52 nm (Band 1), 70 nm (Band 2), and 82 nm (Band 3). Glucobrassicin, glucoraphanin and neoglucobrassicin were found mostly concentrated in Band 1. BDVs affected the metabolic activity of different cancer cell lines in a dose dependent manner compared with untreated cells. Overall, Band 2 and 3 were more toxic than Band 1 irrespective of the cell lines. BDVs were taken up by cells in a dose- and time-dependent manner. Pre-incubation of cells with BDVs resulted in a significant decrease in ROS production in Caco-2 and NCI-H441 stimulated with hydrogen peroxide and SHSY5Y treated with 6-hydroxydopamine, with all three Bands. Our findings open to the possibility to find a novel "green" approach for cancer treatment, focused on using vesicles from broccoli, although a more in-depth characterization of bioactive molecules is warranted.

Discovery of marker peptides of spirulina microalga proteins for allergen detection in processed foodstuffs.

Spirulina (Arthrospira platensis) proteins were extracted, digested, and analyzed by LC-ESI-FTMS/MS to find highly conserved peptides as markers of the microalga occurrence in foodstuffs. Putative markers were firstly chosen after in silico digestion of allergenic proteins, according to the FAO and WHO criteria, after assuring their presence in food supplements and in (un)processed foodsuffs. Parameters such as sensitivity, sequence size, and uniqueness for spirulina proteins were also evaluated. Three peptides belonging to C-phycocyanin beta subunit (P72508) were designated as qualifiers (ETYLALGTPGSSVAVGVGK and YVTYAVFAGDASVLEDR) and quantifier (ITSNASTIVSNAAR) marker peptides and used to validate the method for linearity, recovery, reproducibility, matrix effects, processing effects, LOD, and LOQ. The main aim was to determine spirulina in commercial foodstuffs like pasta, crackers, and homemade bread incurred with the microalga. The possible inclusion of the designated peptides in a standardized method, based on multiple reaction monitoring using a linear ion trap MS, was also demonstrated.

Use of Multivariate Statistics in the Processing of Data on Wine Volatile Compounds Obtained by HS-SPME-GC-MS.

This review takes a snapshot of the main multivariate statistical techniques and methods used to process data on the concentrations of wine volatile molecules extracted by means of solid phase micro-extraction and analyzed using GC-MS. Hypothesis test, exploratory analysis, regression models, and unsupervised and supervised pattern recognition methods are illustrated and discussed. Several applications in the wine volatolomic sector are described to highlight different interactions among the various matrix components and volatiles. In addition, the use of Artificial Intelligence-based methods is discussed as an innovative class of methods for validating wine varietal authenticity and geographical traceability.

Positional Assignment of C-C Double Bonds in Fatty Acyl Chains of Intact Arsenosugar Phospholipids Occurring in Seaweed Extracts by Epoxidation Reactions.

Water-soluble diacyl arsenosugar phospholipids (As-PL) are natural products widespread in marine animals and algae, including the brown alga Undaria pinnatifida, also known as wakame. The systematic recognition of As-PL has been hampered by the lack of standard and of qualitative methods to establish the carbon-carbon double bond positions of unsaturated fatty acyl chains. Here, the epoxidation reaction of fatty acyl substituents of As-PL was carried out with high selectivity by meta-chloroperoxybenzoic acid and the C-C double bond localization was established by collision-induced dissociation of epoxidized species as deprotonated molecules, [epoM - H]-. Reversed-phase liquid chromatography (RPLC) separation and a sequential triple-stage MS (i.e., MS3) analysis of unsaturated and epoxidized As-PL were very helpful to characterize the carbon-carbon double bond locations of both sn-1 and sn-2 fatty acyl chains, starting from a diagnostic product ion pair with 16.0 Da mass difference. These results indicate that intact As-PL can be annotated in terms of fatty acyl chain composition and in terms of their C-C double bond position(s). Interestingly, hexadecenoic (16:1 Δ9) and octadecenoic (18:1 Δ9) along with octadecadienoic (18:2 Δ9,12) and octadecatrienoic (18:3 Δ9,12,15) were found to be the most abundant unsaturated fatty acyl chains of As-PL in the brown alga wakame, thus confirming it as a good source of essential fatty acids with a balanced ω6/ω3 ratio. Although the toxicity of As-including metabolites of algal As-PL is still a matter of debate and needs to be studied in more detail, the described approach can be exploited to assess if As-PL could contribute to the supply of essential fatty acids related to the use of algae as nutritious food.

Tree Nuts and Peanuts as a Source of Beneficial Compounds and a Threat for Allergic Consumers: Overview on Methods for Their Detection in Complex Food Products.

Consumption of tree nuts and peanuts has considerably increased over the last decades due to their nutritional composition and the content of beneficial compounds. On the other hand, such widespread consumption worldwide has also generated a growing incidence of allergy in the sensitive population. Allergy to nuts and peanuts represents a global relevant problem, especially due to the risk of the ingestion of hidden allergens as a result of cross-contamination between production lines at industrial level occurring during food manufacturing. The present review provides insights on peanuts, almonds, and four nut allergens-namely hazelnuts, walnuts, cashew, and pistachios-that are likely to cross-contaminate different food commodities. The paper aims at covering both the biochemical aspect linked to the identified allergenic proteins for each allergen category and the different methodological approaches developed for allergens detection and identification. Attention has been also paid to mass spectrometry methods and to current efforts of the scientific community to identify a harmonized approach for allergens quantification through the detection of allergen markers.

Glycerophospholipidomics of Five Edible Oleaginous Microgreens.

Microgreens are a special type of vegetal product, born as a culinary novelty (traditionally used to garnish gourmet dishes) and then progressively studied for their potentially high content in nutraceuticals, like polyphenolic compounds, carotenoids, and glucosinolates, also in the perspective of implementing their cultivation in space stations/colonies. Among further potential nutraceuticals of microgreens, lipids have received very limited attention so far. Here, glycerophospholipids contained in microgreens of typical oleaginous plants, namely, soybean, chia, flax, sunflower, and rapeseed, were studied using hydrophilic interaction liquid chromatography (HILIC), coupled to high-resolution Fourier transform mass spectrometry (FTMS) or low-resolution collisionally induced dissociation tandem mass spectrometry (CID-MS2) with electrospray ionization (ESI). Specifically, this approach was employed to obtain qualitative and quantitative profiling of the four main classes of glycerophospholipids (GPL) found in the five microgreens, i.e., phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidylglycerols (PG), and phosphatidylinositols (PI). Saturated chains with 16 and 18 carbon atoms and unsaturated 18:X (with X = 1-3) chains emerged as the most common fatty acyl substituents of those GPL; a characteristic 16:1 chain (including a C═C bond between carbon atoms 3 and 4) was also found in some PG species. Among polyunsaturated acyl chains, the 18:3 one, likely referred mainly to α-linolenic acid, exhibited a relevant incidence, with the highest estimated amount (corresponding to 160 mg per 100 g of lyophilized vegetal tissue) found for chia. This outcome opens interesting perspectives for the use of oleaginous microgreens as additional sources of essential fatty acids, especially in vegetarian/vegan diets.

A new paradigm to search for allergenic proteins in novel foods by integrating proteomics analysis and in silico sequence homology prediction: Focus on spirulina and chlorella microalgae.

Since novel nutrient sources with high protein content, such as yeast, fungi, bacteria, algae, and insects, are increasingly introduced in the consumer market, safety evaluation studies on their potentially allergenic proteins are required. A pipeline for in silico establishing the sequence-based homology between proteins of spirulina (Arthrospira platensis) and chlorella (Chlorella vulgaris) micro-algae and those included in the AllergenOnline (AO) database (AllergenOnline.org) is described. The extracted proteins were first identified through tryptic peptides analysis by reversed-phase liquid chromatography and high resolution/accuracy Fourier-transform tandem mass spectrometry (RPLC-ESI-FTMS/MS), followed by a quest on the UniProt database. The AO database was subsequently interrogated to assess sequence similarity between identified microalgal proteins and known allergens, based on criteria established by the World Health Organization (WHO) and Food and Agriculture Organization (FAO). A direct search for microalgal proteins already included in allergen databases was also performed using the Allergome database. Six proteins exhibiting a significant homology with food allergens were identified in spirulina extracts. Five of them, i.e., two thioredoxins (D4ZSU6, K1VP15), a superoxide dismutase (C3V3P3), a glyceraldehyde-3-phosphate dehydrogenase (K1W168), and a triosephosphate isomerase (D5A635), resulted from the search on AO. The sixth protein, C-phycocyanin beta subunit (P72508), was directly obtained after examining the Allergome database. Two proteins exhibiting significant sequence homology with food allergens were retrieved in chlorella extracts, viz. calmodulin (A0A2P6TFR8), which is related to troponin c (D7F1Q2), and fructose-bisphosphate aldolase (A0A2P6TDD0). Specific serum screenings based on immunochemical tests should be undertaken to confirm or rule out the allergenicity of the identified proteins.

Exploring the Isomeric Precursors of Olive Oil Major Secoiridoids: An Insight into Olive Leaves and Drupes by Liquid-Chromatography and Fourier-Transform Tandem Mass Spectrometry.

Secoiridoids play a key role in determining health benefits related to a regular consumption of extra-virgin olive oil (EVOO), in which they are generated from precursors of the same class naturally occurring in drupes and leaves of the olive (Olea europaea L.) plant. Here, reversed-phase liquid chromatography coupled to electrospray ionization and Fourier-transform single/tandem mass spectrometry (RPLC-ESI-FTMS and MS/MS) was employed for a structural elucidation of those precursors. The presence of three isoforms in both matrices was assessed for oleuropein ([M-H]- ion with m/z 539.1770) and was emphasized, for the first time, also for ligstroside (m/z 523.1821) and for the demethylated counterparts of the two compounds (m/z 525.1614 and 509.1665, respectively). However, only the prevailing isoform included an exocyclic double bond between carbon atoms C8 and C9, typical of oleuropein and ligstroside; the remaining, less abundant, isoforms included a C=C bond between C8 and C10. The same structural difference was also observed between secoiridoids named elenolic acid glucoside and secoxyloganin (m/z 403.1246). This study strengthens the hypothesis that secoiridoids including a C8=C10 bond, recently recognized as relevant species in EVOO extracts, arise mainly from specific enzymatic/chemical transformations occurring on major oleuropein/ligstroside-like precursors during EVOO production, rather than from precursors having that structural feature.

HILIC-ESI-MS analysis of phosphatidic acid methyl esters artificially generated during lipid extraction from microgreen crops.

The uncontrolled activation of endogenous enzymes may introduce both qualitative and quantitative artefacts when lipids are extracted from vegetal matrices. In the present study, a method based on hydrophilic interaction liquid chromatography coupled either to high-resolution/accuracy Fourier-transform mass spectrometry (HILIC-ESI-FTMS) or to linear ion trap multiple stage mass spectrometry (HILIC-ESI-MSn , with n = 2 and 3) with electrospray ionization was developed to unveil one of those artefacts. Specifically, the artificial generation of methyl esters of phosphatidic acids (MPA), catalysed by endogenous phospholipase D (PLD) during lipid extraction from five oleaginous microgreen crops (chia, soy, flax, sunflower and rapeseed), was studied. Phosphatidylcholines (PC) and phosphatidylglycerols (PG) were found to be the most relevant precursors of MPA among glycerophospholipids (GPLs), being involved in a transphosphatidylation process catalysed by PLD and having methanol as a coreactant. The combination of MS2 and MS3 measurements enabled the unambiguous recognition of MPA from their fragmentation pathways, leading to distinguish them from isobaric PA including a further CH2 group on their side chains. PLD was also found to catalyse the hydrolysis of PC and PG to phosphatidic acids (PAs). The described transformations were confirmed by the remarkable decrease of MPA abundance observed when isopropanol, known to inhibit PLD, was tentatively adopted instead of water during the homogenization of microgreens. The unequivocal identification of MPA might be exploited to assess if GPL alterations are actually triggered by endogenous PLD during lipid extractions from specific vegetal tissues.