Mass spectrometric characterization of aminophospholipids containing N-(2-hydroxyethyl)glycine in kombu algae extracts.

RATIONALE: 1,2-Diacyl-sn-glycero-3-phospho-O-[N-(2-hydroxyethyl)glycines] (PHEGs) are a class of rare aminophospholipids found specifically in brown algae, including kombu seaweed. Despite their potential importance in algal physiology, a comprehensive mass spectrometry (MS) characterization, useful to understand their biological behaviour, is still lacking. METHODS: To establish the structural regiochemical features of PHEGs, we employed hydrophilic interaction liquid chromatography (HILIC). Following separation, the isolated band of PHEGs was analyzed using MS techniques. This included multistage tandem MS experiments, performed in both positive and negative electrospray ionization modes at low and high resolution. RESULTS: By comparing MS/MS and MS3 spectra acquired in negative ion mode, the regiochemical rules for PHEG identification were established. The most abundant PHEG species in kombu seaweed, from both Laminaria ochroleuca (European Atlantic) and Laminaria longissima (Japan), was identified as PHEG 20:4/20:4. Less abundant species included PHEG 20:4/20:5 and hydroxylated forms of both PHEG 20:4/20:4 (i.e. 40:8;O) and 20:4/20:5 (40:9;O). The presence of a lyso PHEG 20:4 was consistently detected but at very low levels. CONCLUSIONS: This study employed MS analysis to elucidate the regiochemical patterns of PHEGs in kombu seaweed. We identified PHEG 20:4/20:4 as the dominant species, along with several less abundant variants, including hydroxylated forms. These findings provide valuable insights into the potential roles and metabolism of PHEGs in brown algae, paving the way for further investigation into their biological functions.

Proteomics for Microalgae Extracts by High-Resolution Mass Spectrometry.

Two protocols of protein extraction from Arthrospira platensis (spirulina) microalgae to study their proteome by mass spectrometry (MS) are here presented. The first is based on an aqueous buffer solution of Tris-HCl and the second on cold acetone. The identification of proteins was carried out by a bottom-up approach, which involves enzymatic digestion of extracted proteins followed by either matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) MS or liquid chromatography (LC) coupled with electrospray ionization (ESI) and Fourier-transform tandem MS. While MALDI-TOF MS allowed for a fast peptide mass fingerprinting (PMF) check yet identifying less than 20 proteins in the extracted samples, the data-dependent acquisitions (DDA) mode of reversed-phase (RP) LC-ESI tandem FTMS/MS separations allowed us to recognize more than one hundred proteins by searching into dedicated spectral libraries. The application of MALDI-TOF MS analysis was found, however, of great support for preliminary investigations of cyanobacteria samples before proceeding with the RPLC-ESI-MS/MS DDA investigation, which definitively allows for a qualitative proteome analysis also of minor spirulina proteins in processed foodstuffs. Although the protein content in spirulina can be influenced by cultivation and environmental conditions, e.g., light intensity, climate, and water/air quality, here the qualitative chemical profiles of the examined samples were characterized by similar composition in high-quality proteins as phycocyanins and phycoerythrins.

Development of a New Binary Matrix for the Comprehensive Analysis of Lipids and Pigments in Micro- and Macroalgae Using MALDI-ToF/ToF Mass Spectrometry.

While edible algae might seem low in fat, the lipids they contain are crucial for good health and preventing chronic diseases. This study introduces a binary matrix to analyze all the polar lipids in both macroalgae (Wakame-Undaria pinnatifida, Dulse-Palmaria palmata, and Nori-Porphyra spp.) and microalgae (Spirulina-Arthrospira platensis, and Chlorella-Chlorella vulgaris) using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The key lies in a new dual matrix made by combining equimolar amounts of 1,5-diaminonaphthalene (DAN) and 9-aminoacridine (9AA). This combination solves the limitations of single matrices: 9AA is suitable for sulfur-containing lipids and acidic phospholipids, while DAN excels as an electron-transfer secondary reaction matrix for intact chlorophylls and their derivatives. By employing the equimolar binary matrix, a wider range of algal lipids, including free fatty acids, phospholipids, glycolipids, pigments, and even rare arsenosugarphospholipids were successfully detected, overcoming drawbacks related to ion suppression from readily ionizable lipids. The resulting mass spectra exhibited a good signal-to-noise ratio at a lower laser fluence and minimized background noise. This improvement stems from the binary matrix's ability to mitigate in-source decay effects, a phenomenon often encountered for certain matrices. Consequently, the data obtained are more reliable, facilitating a faster and more comprehensive exploration of algal lipidomes using high-throughput MALDI-MS/MS analysis.

Lipidomics reveals the reshaping of the mitochondrial phospholipid profile in cells lacking OPA1 and mitofusins.

Depletion or mutations of key proteins for mitochondrial fusion, like optic atrophy 1 (OPA1) and mitofusins 1 and 2 (Mfn 1 and 2), are known to significantly impact the mitochondrial ultrastructure, suggesting alterations of their membranes' lipid profiles. In order to make an insight into this issue, we used hydrophilic interaction liquid chromatography coupled with electrospray ionization-high resolution MS to investigate the mitochondrial phospholipid (PL) profile of mouse embryonic fibroblasts knocked out for OPA1 and Mfn1/2 genes. One hundred sixty-seven different sum compositions were recognized for the four major PL classes of mitochondria, namely phosphatidylcholines (PCs, 63), phosphatidylethanolamines (55), phosphatidylinositols (21), and cardiolipins (28). A slight decrease in the cardiolipin/PC ratio was found for Mfn1/2-knockout mitochondria. Principal component analysis and hierarchical cluster analysis were subsequently used to further process hydrophilic interaction liquid chromatography-ESI-MS data. A progressive decrease in the incidence of alk(en)yl/acyl species in PC and phosphatidylethanolamine classes and a general increase in the incidence of unsaturated acyl chains across all the investigated PL classes was inferred in OPA1 and Mfn1/2 knockouts compared to WT mouse embryonic fibroblasts. These findings suggest a reshaping of the PL profile consistent with the changes observed in the mitochondrial ultrastructure when fusion proteins are absent. Based on the existing knowledge on the metabolism of mitochondrial phospholipids, we propose that fusion proteins, especially Mfns, might influence the PL transfer between the mitochondria and the endoplasmic reticulum, likely in the context of mitochondria-associated membranes.

Exploration of the Lipid Profile of Edible Oleaginous Microgreens by Mass Spectrometry-Based Lipidomics.

The spreading awareness of the health benefits associated with the consumption of plant-based foods is fueling the market of innovative vegetable products, including microgreens, recognized as a promising source of bioactive compounds. To evaluate the potential of oleaginous plant microgreens as a source of bioactive fatty acids, gas chromatography-mass spectrometry was exploited to characterize the total fatty acid content of five microgreens, namely, chia, flax, soy, sunflower, and rapeseed (canola). Chia and flax microgreens appeared as interesting sources of α-linolenic acid (ALA), with total concentrations of 2.6 and 2.9 g/100 g of dried weight (DW), respectively. Based on these amounts, approximately 15% of the ALA daily intake recommended by the European Food Safety Authority can be provided by 100 g of the corresponding fresh products. Flow injection analysis with high-resolution Fourier transform single and tandem mass spectrometry enabled a semi-quantitative profiling of triacylglycerols (TGs) and sterol esters (SEs) in the examined microgreen crops, confirming their role as additional sources of fatty acids like ALA and linoleic acid (LA), along with glycerophospholipids. The highest amounts of TGs and SEs were observed in rapeseed and sunflower microgreens (ca. 50 and 4-5 µmol/g of DW, respectively), followed by flax (ca. 20 and 3 µmol/g DW). TG 54:9, 54:8, and 54:7 prevailed in the case of flax and chia, whereas TG 54:3, 54:4, and 54:5 were the most abundant TGs in the case of rapeseed. ß-Sitosteryl linoleate and linolenate were generally prevailing in the SE profiles, although campesteryl oleate, linoleate, and linolenate exhibited a comparable amount in the case of rapeseed microgreens.

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.

The Effect of Milling on the Ethanolic Extract Composition of Dried Walnut (Juglans regia L.) Shells.

This study investigates the ethanolic extract of dried walnut (Juglans regia L.) shells upon hammer milling (HM) and ball milling (BM) grinding processes. Marked differences were observed in the attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectra. The two extracts were investigated by reversed-phase liquid chromatography coupled with electrospray ionization and high-resolution mass spectrometry (RPLC-ESI-HRMS). Following enzymatic digestion, the fatty acids (FAs) were examined, and tandem MS of epoxidized species was applied to establish the C-C double bond position; the most abundant species were FA 18:2 Δ9,12, FA 18:1 Δ9, and FA 18:3 Δ9,12,15. However, no significant qualitative differences were observed between FAs in the two samples. Thus, the presence of potential active secondary metabolites was explored, and more than 30 phenolic compounds, including phenols, ellagic acid derivatives, and flavonoids, were found. Interestingly, the HM samples showed a high concentration of ellagitannins and hydrolyzable tannins, which were absent in the BM sample. These findings corroborate the greater phenolic content in the HM sample, as evaluated by the Folin-Ciocalteu test. Among the others, the occurrence of lanceoloside A at m/z 391.1037 [C19H20O9-H]-, and a closely related benzoyl derivate at m/z 405.1190 (C20H22O9-H]-), was ascertained. The study provides valuable information that highlights the significance of physical pre-treatments, such as mill grinding, in shaping the composition of extracts, with potential applications in the biorefinery or pharmaceutical industries.

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.

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.

Oxidative NHC Catalysis: An Unconventional Tool for Macrocyclic Oligoesters Synthesis.

The application of N-heterocyclic carbene (NHC) catalysis under highly diluted oxidative condition to the polycondensation of dialdehydes and diols is herein presented as an alternative, atom-economical synthetic route to macrocyclic oligoesters (MCOs). The disclosed protocol paves the way to the straightforward access to MCOs, starting from commercial dialdehydes, avoiding the use of toxic diacyl chlorides, commonly employed in traditional MCOs synthetic processes. The method is totally metal-free, takes place in the green Me-THF solvent and requires the use of a fully recyclable quinone oxidant. The protocol versatility is confirmed by the employment of fossil-based and bio-based monomers such as 2,5-diformylfuran (DFF), 2,5-bis(hydroxymethyl)furan (BHMF), and isomannide, synthesizing a series of novel and known synthetically relevant macrocyclic oligoesters, fully characterized by NMR and MALDI-TOF MS analysis, with product yields (51-86 %) comparable to those obtained by traditional synthetic routes. Finally, to emphasize the synthetic relevance of the target macrocycles, an entropically-driven ring opening polymerization (ED-ROP) key study has been performed, optimizing the organocatalyzed synthesis of poly(2,5-furan-dimethylene 2,5 furandicarboxylate) (PBHMF) with number-average molecular weight up to 8200 g mol-1 and 66 % isolated yield.

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.

The Mycotoxin Patulin Inhibits the Mitochondrial Carnitine/Acylcarnitine Carrier (SLC25A20) by Interaction with Cys136 Implications for Human Health.

The effect of mycotoxin patulin (4-hydroxy-4H-furo [3,2c] pyran-2 [6H] -one) on the mitochondrial carnitine/acylcarnitine carrier (CAC, SLC25A20) was investigated. Transport function was measured as [3H]-carnitineex/carnitinein antiport in proteoliposomes reconstituted with the native protein extracted from rat liver mitochondria or with the recombinant CAC over-expressed in E. coli. Patulin (PAT) inhibited both the mitochondrial native and recombinant transporters. The inhibition was not reversed by physiological and sulfhydryl-reducing reagents, such as glutathione (GSH) or dithioerythritol (DTE). The IC50 derived from the dose-response analysis indicated that PAT inhibition was in the range of 50 µM both on the native and on rat and human recombinant protein. The kinetics process revealed a competitive type of inhibition. A substrate protection experiment confirmed that the interaction of PAT with the protein occurred within a protein region, including the substrate-binding area. The mechanism of inhibition was identified using the site-directed mutagenesis of CAC. No inhibition was observed on Cys mutants in which only the C136 residue was mutated. Mass spectrometry studies and in silico molecular modeling analysis corroborated the outcomes derived from the biochemical assays.

Analysis of surfactants by mass spectrometry: Coming to grips with their diversity.

Surfactants are surface-active agents widely used in numerous applications in our daily lives as personal care products, domestic, and industrial detergents. To determine complex mixtures of surfactants and their degradation products, unselective and rather insensitive methods, based on colorimetric and complexometric analyses are no longer employable. Analytical methodologies able to determine low concentration levels of surfactants and closely related compounds in complex matrices are required. The recent introduction of robust, sensitive, and selective mass spectrometry (MS) techniques has led to the rapid expansion of the surfactant research field including complex mixtures of isomers, oligomers, and homologues of surfactants as well as their chemically and biodegradation products at trace levels. In this review, emphasis is given to the state-of-the-art MS-based analysis of surfactants and their degradation products with an overview of the current research landscape from traditional methods involving hyphenate techniques (gas chromatography-MS and liquid chromatography-MS) to the most innovative approaches, based on high-resolution MS. Finally, we outline a detailed explanation on the utilization of MS for mechanistic purposes, such as the study of micelle formation in different solvents.

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.

Inhibition of the carnitine acylcarnitine carrier by carbon monoxide reveals a novel mechanism of action with non-metal-containing proteins.

Both toxic and physiological effects of CO are mostly caused by well described interactions with heme-groups of proteins. Interactions of CO with non-heme proteins have also been unveiled. Besides interaction of CO with mitochondrial heme containing respiratory complexes, a BK channel and the phosphate carrier which do not contain metal cofactors, have been identified as CO targets. However, the molecular mechanisms of interaction with non-metal-containing proteins are not understood. We show in this work the effect of CO on the mitochondrial carnitine carrier (SLC25A20) using CORM-3, a widely recognized CO releasing compound. CO exerts an inhibitory effect at the micromolar concentration on the transport function of the transporter extracted from treated mitochondria. The effect is due to a single Cys residue, C136 as revealed by mass spectrometry analysis. A computational approach predicted the need for vicinal Asp and Lys residues for the C136 carbonylation to occur. These data demonstrate a novel mechanism of interaction of CO with a protein not containing metal atoms and will enable the prediction of CO targets.

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.

Synthesis and Investigation of Novel CHCA-Derived Matrices for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Analysis of Lipids.

A significant area of study and upgrading for increasing sensitivity and general performances of matrix-assisted laser-desorption ionization (MALDI) mass spectrometry (MS) is related to matrix design. Several efforts have been made to address the challenge of low-mass-region interference-free for metabolomics analysis and specifically for lipidomics. To this aim, rationally designed matrices as 4-chloro-α-cyanocinnamic acid (ClCCA) were introduced and reported to provide enhanced analytical performances. We have taken this rational design one step further by developing and optimizing new MALDI matrices with a range of modifications on the CHCA core, involving different functionalities and substituents. Of particular interest was the understanding of the electron-withdrawing (e.g., nitro-) or donating (e.g., methoxy-) effects along with the extent of conjugation on the ionization efficiency. In the present work, ten matrices were designed on a reasonable basis, synthesized, and characterized by NMR and UV spectroscopies and laser desorption ionization. With the assistance of these putative MALDI matrices, samples containing phospholipids (PL), and neutral di-/tri-acylglycerols (DAG, TAG) were investigated using milk, fish, blood, and human plasma extracts. In comparison with CHCA and ClCCA, four of them, viz. [(2E,4E)-2-cyano-5-(4-methoxyphenyl)penta-2,4-dienoic acid] (1), [(2E,4E)-2-cyano-5-(4-nitrophenyl)penta-2,4-dienoic acid] (2), [(E)-2-cyano-3-(6-methoxynaphthalen-2-yl)acrylic acid] (6) and [(E)-2-cyano-3-(naphthalen-2-yl)acrylic acid] (7) displayed good to even excellent performances as MALDI matrices in terms of ionization capability, interference-free spectra, S/N ratio, and reproducibility. Especially compound 7 (cyano naphthyl acrylic acid, CNAA) was the election matrix for PL analysis and matrix 2 (cyano nitrophenyl dienoic acid, CNDA) for neutral lipids such as DAG and TAG in positive ion mode.