1,720,970 research outputs found
Proteomics for Microalgae Extracts by High-Resolution Mass Spectrometry
No full textTwo 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
Discovery of marker peptides of spirulina microalga proteins for allergen detection in processed foodstuffs
Get PDFSpirulina (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
1,5-Diaminonaphtalene is a Highly Performing Electron-Transfer Secondary-Reaction Matrix for Laser Desorption Ionization Mass Spectrometry of Indolenine-Based Croconaines
Get PDFCroconaine dyes are appealing molecules synthesized via the condensation of croconic acid and reactive electron-donating aromatic or heterocyclic systems. Here, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) investigation of indolenine-based croconaines is presented for the first time. Archetype proton-transfer matrices, such as 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA), 9-aminoacridine (9AA) as the protonating/deprotonating matrix, and electron-transfer (ET) secondary-reaction matrices, such as 1,5-diaminonapthalene (DAN) and trans-2-[3-(4-t-butyl-phenyl)-2-methyl-2-propenylidene]malononitrile (DCTB), were investigated. DHB, CHCA, and 9AA generate a mix of odd-electron molecular ions and protonated, sodiated, and potassiated adducts. Among the ET matrices, DAN was found to be capable of directing the ionization process toward the exclusive formation of odd-electron molecular ions M+• without fragmentation. MALDI tandem MS provides useful structural characterization of croconaine dyes, thus making identification very straightforward for all investigated compounds. Interestingly the fragmentation of bromo-containing croconaines revealed, for the first time, the gas-phase formation of a bromime cation [Br]+
Strategies for the analysis of arsenolipids in marine foods: A review
No full text: 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
Untargeted lipidomics for evaluating fish authenticity: the case of wild-caught and farmed species of Sparus aurata
Get PDFAim:
This study aims to develop sensitive and reliable analytical technologies to enable the distinction between wild-caught and farmed fish through appropriate molecular markers to protect consumers from fraudulent fish labelling. Gilthead sea bream, Sparus aurata L. (S. aurata L.), is a very common fish used as foodstuff worldwide and globally produced in aquaculture in the Mediterranean basin. Wild-caught and farmed species are very different in feed and lifestyle and the quality and safety of these products strongly depend on fish growth, processing history, and storage conditions.
Methods:
Hydrophilic interaction liquid chromatography (HILIC) coupled with electrospray ionization (ESI) and Fourier-transform mass spectrometry (FTMS; HILIC-ESI-FTMS) was employed to discriminate the phospholipidome profiles of fillets extracts of wild-caught from farmed gilthead sea breams.
Results:
The untargeted approach led to the annotation of a total of 216 phospholipids (PLs), namely 65 phosphatidylethanolamines (PEs), 27 lyso-PEs (LPEs), 61 phosphatidylcholines (PCs), 34 lyso-PCs (LPCs), and 29 sphingomyelins (SMs). Untargeted lipidomics data were investigated by principal component analysis (PCA) and K-means clustering. Lyso-PLs (LPLs) of PEs and PCs including ether-linked side chains were found as discriminating markers between the two types of fish samples. The PLs that were most responsible for distinguishing between the lipid extracts of farmed and wild S. aurata fillets were successfully characterized by tandem mass spectrometry (MS/MS). The analysis revealed that wild fillet lipid extracts contained some PE exhibiting ether bonds (PE P-), viz. 16:0, 18:0, 18:1, and 18:2 and polyunsaturated fatty acyl chains (i.e., 22:6 and 22:5). In farmed species, the estimated abundance ratios of fatty acyl chains 20:4/18:2 and 22:6/20:5 were 0.9 and 0.05, respectively. However, in wild-caught fish, these ratios were found to be two-fold higher and four-fold higher, respectively.
Conclusions:
This work demonstrates that the combination of HILIC-ESI-FTMS and chemometrics can serve as a valuable tool for evaluating fish authenticity and assessing quality concerns by monitoring specific lipid ratios
HILIC‐ESI‐MS analysis of phosphatidic acid methyl esters artificially generated during lipid extraction from microgreen crops
Get PDFThe 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‐MS( n ), 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 MS(2) and MS(3) measurements enabled the unambiguous recognition of MPA from their fragmentation pathways, leading to distinguish them from isobaric PA including a further CH(2) 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
Ultrasound-assisted protein extraction for deep proteome analysis of Spirulina and Chlorella microalgae
Get PDFMicroalgae Spirulina (Arthrospira platensis) and Chlorella (Chlorella vulgaris) are gaining attention as sustainable alternative protein sources to address the needs of a growing global population. However, their tough cell walls present a significant challenge for efficient and more quantitative protein extraction. This study introduces a simple, cost-effective, and scalable “one-pot” protocol for extracting microalgal proteins by ultrasound-assisted methods in an aqueous buffer. The combination of ultrasonication and sodium deoxycholate successfully breaks down the resilient cell walls, improves protein solubility in water, and achieves high extraction yields of 81–86 %. This performance significantly surpasses traditional methods, such as Tris-HCl buffer (0.7–1.2 % yields) and chloroform/methanol solvent (4.5–14 % yields). Using this method, 1354 proteins were identified in Spirulina and 771 in Chlorella, each with over 10 % sequence coverage. This enabled the recovery of numerous bioactive peptides, including 161 candidates for Spirulina and 60 for Chlorella from phycobiliproteins, all comprising at least three amino acid residues. These peptides hold the potential for creating bioactive hydrolysates and functional ingredients for food and pharmaceutical applications, following detergent removal via appropriate purification. This optimized protocol offers a promising solution for large-scale, safe microalgae processing and facilitates the production of bioavailable peptides from microalgae species.</p
Oxidative NHC Catalysis: An Unconventional Tool for Macrocyclic Oligoesters Synthesis
No full textThe 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
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