Search

Glucuronic acid containing diacylglycerols (3-(O-α-d-glucuronopyranosyl)-1,2-diacyl-sn-glycerols, GlcA-DAG) are glycolipids of plant membranes especially formed under phosphate-depletion conditions. An analytical approach for the structural characterization of GlcA-DAG in red ripe tomato (Solanum lycopersicum L.) extracts, based on reversed-phase liquid chromatography (RPLC) coupled with electrospray ionization (ESI) and tandem mass spectrometry (MS/MS) using a linear ion trap, is described in this paper. At least 14 GlcA-DAG (R1/R2) species, including four regioisomers, containing three predominant fatty acyl chains C16:0, C18:2, and C18:3, were identified for the first time. Moreover, 29 GlcA-DAG acylated on the glucuronosyl ring (acyl-R3 GlcA-DAG) were discovered, alongside 15 acylated lyso-forms, i.e., acylated 3-(O-α-d-glucuronosyl)monoacylglycerols, abbreviated as acyl-R3 GlcA-MAG (R1/0) or (0/R2). Although many of these acylated lyso-forms were isomeric with GlcA-DAG (i.e., acyl chains with equivalent sum composition), they were successfully separated by reversed-phase liquid chromatography (RPLC) using a solid-core C18 column packed with 2.6 μm particle size. Tandem MS (and eventually MS3) data obtained from sodium adducts ([M + Na]+) and deprotonated molecules ([M - H]-) were fundamental to detect diagnostic product ions related to the glucuronosyl ring and then determine the identity of all investigated glycolipids, especially to recognize the acyl chain linked to the ring. A classification of GlcA-MAG, GlcA-DAG, and acylated GlcA-DAG and GlcA-MAG was generated by an in house-built database. The discovery of acylated derivatives emphasized the already surprising heterogeneity of glucuronic acid-containing mono- and diacylglycerols in tomato plants, stimulating interesting questions on the role played by these glycolipids.

1,2-diacyl-sn-glycero-3-phospho-N-acyl-ethanolamines (NAPE) are low abundance phospholipids but important constituents of intracellular membranes of plant tissues, responsible to generate bioactive...

The chemical identity of arsenosugar phospholipids (As-PL) as mono- (i.e., lyso, L-As-PL) and diacyl-arsenosugar PL in four edible and common marine alga samples, such as nori (Porphyra spp.), waka...

The lipidome of a brown seaweed commonly known as wakame (Undaria pinnatifida), which is grown and consumed around the world, including Western countries, as a healthy nutraceutical food or supplement, was here extensively examined. The study was focused on the characterization of phospholipids (PL) and glycolipids (GL) by liquid chromatography (LC), either hydrophilic interaction LC (HILIC) or reversed-phase LC (RPLC), coupled to electrospray ionization (ESI) and mass spectrometry (MS), operated both in high and in low-resolution mode. Through the acquisition of single (MS) and tandem (MS/MS) mass spectra more than 200 PL and GL of U. pinnatifida extracts were characterized in terms of lipid class, fatty acyl (FA) chain composition (length and number of unsaturations), and regiochemistry, namely 16 SQDG, 6 SQMG, 12 DGDG, 5 DGMG, 29 PG, 8 LPG, 19 PI, 14 PA, 19 PE, 8 PE, 38 PC, and 27 LPC. The FA (C16:0) was the most abundant saturated acyl chain, whereas the monounsaturated C18:1 and the polyunsaturated C18:2 and C20:4 chains were the prevailing ones. Odd-numbered acyl chains, iJ., C15:0, C17:0, C19:0, and C19:1, were also recognized. While SQDG exhibited the longest and most unsaturated acyl chains, C18:1, C18:2, and C18:3, in the sn-1 position of glycerol, they were preferentially located in the sn-2 position in the case of PL. The developed analytical approach might pave the way to extend lipidomic investigations also for other edible marine algae, thus emphasizing their potential role as a source of bioactive lipids.

RATIONALE Several bioactive compounds, including phenolic acids and secoiridoids, are transferred from olive drupes to olive oil during the first stage of production. Here, the characterization of these low molecular weight (LMW) compounds in olive oil and in closely related processing materials, like olive leaves (OL) and olive mill wastewaters (OMW), was faced up, for the first time, by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS). METHODS A novel binary matrix composed of 1,8-bis(tetramethylguanidino)naphthalene (TMGN) and 9-aminoacridine (9AA) (1:1 molar ratio), displaying excellent ionization properties at low levels of laser energy, was employed in reflectron negative ion mode by a MALDI TOF/TOF system equipped with a neodymium-doped yttrium lithium fluoride (Nd:YLF) laser (345 nm). MS/MS experiments were performed by using ambient air as the collision gas. RESULTS Four major secoiridoids typically present in olive oil, i.e., the aglycones of oleuropein and ligstroside, and oleacein and olecanthal at m/z 377.1, 361.1, 319.1 and 303.1, respectively, were detected in virgin olive oil (VOO) extracts, along with some of their chemical/enzymatic hydrolysis by-products, such as elenolic (m/z 241.1), decarboxymethyl-elenolic acids (m/z 183.1) and hydroxytyrosol (m/z 153.1). Besides oleuropein aglycone and oleacein, hydroxylated derivatives of decarboxymethyl-elenolic acid and hydroxytyrosol were evidenced in OMW. CONCLUSIONS While oleuropein was confirmed in OL extracts, several interesting phenolic compounds, including hydroxytyrosol, were recognized in OMW. The proposed approach based on the use of a novel binary matrix for MALDI MS/MS analyses of LMW bioactive compounds can be considered a promising analytical tool for a rapid screening of the phenolic fraction in olive oils and related processing wastes.

Several mobile phase additives (i.e., organic acids and their ammonium salts) were used to modulate the chromatographic retention of cyanocobalamin and its cis-diaminemonochloroplatinum(II) conjugate, depending on the specific nature of the stationary phase. Regardless of the mobile phase additive, the positively charged cyanocobalamin-cis-diaminemonochloroplatinum(II) conjugate was systematically less retained than cyanocobalamin on a conventional octadecyl-silica column. In contrast, the amide-embedded C18 column exhibited a progressive increase in the conjugate retention time upon changing the mobile phase additive from organic (acetic, formic and trifluoroacetic) acids to ammonium salts, ultimately leading to an inversion of the elution order. This change of retention was interpreted by invoking the interplay between hydrophobic interactions, hydrogen bonding between the conjugate and the polar amide groups and the ion-pairing ability of the lyophilic counterions, whereby the acetate anion was found to be the most suitable to control the solute retention.

Rationale Cisplatin (CP) is a widely used anticancer drug characterized by toxic side effects that could be alleviated using novel delivery systems including CP prodrugs. The in vitro incubation of a putative prodrug, obtained from cyanocobalamin (CNCbl) and cis-diamminemonochloroplatinum(II) (mCP), with nucleoside monophosphates (NMPs) was investigated. Methods The in vitro reactions between the putative prodrug CNCbl-mCP and the NMPs of adenosine (AMP), guanosine (GMP), cytidine (CMP) and uridine (UMP) were carried out in slightly acidic water-methanol solutions at 37°C for 24 h. Each sample was examined using reversed-phase liquid chromatography coupled with electrospray ionization in positive ion mode and tandem mass spectrometry (RPLC/ESI-MS/MS) by collision-induced dissociation in a linear ion-trap mass spectrometer. Results Seven adducts were recognized as formed by substitution reactions of the chloride ligand in planar CP. Comparison between observed and theoretical isotopic patterns together with MS/MS fragmentation pathways revealed the presence of single or multiple binding sites depending on the NMP involved. The CNCbl-mCP conjugate was found to interact with N7 or O4 atoms of GMP and UMP, respectively, generating single adducts, while two isomeric adducts were observed for CMP. Finally, AMP gave rise to three isomeric adducts. Conclusions In agreement with literature data relevant to the interaction between CP and NMPs, the most reactive nucleotides were AMP and GMP. The present RPLC/ESI-MS/MS approach is very promising for investigation of the reactions of CP conjugates with ribonucleotides not only in vitro but also in vivo.

Lipidomics suffers from the lack of fast and reproducible tools to obtain both structural information on intact phospholipids (PL) and fatty acyl chain composition. Hydrophilic interaction liquid chromatography with electrospray ionization coupled to an orbital-trap Fourier-transform analyzer operating using all ion fragmentation mode (HILIC-ESI-FTMS-AIF MS) is seemingly a valuable resource in this respect. Here, accurate m/z values, HILIC retention times and AIF MS scan data were combined for PL assignment in standard mixtures or real lipid extracts. AIF scans in both positive and negative ESI mode, achieved using collisional induced dissociation for fragmentation, were applied to identify both the head-group of each PL class and the fatty acyl chains, respectively. An advantage of the AIF approach was the concurrent collection of tandem MS-like data, enabling the identification of linked fatty acyl chains of precursor phospholipids through the corresponding carboxylate anions. To illustrate the ability of AIF in the field of lipidomics, two different types of real samples, i.e., the lipid extracts obtained from human plasma and dermal fibroblasts, were examined. Using AIF scans, a total of 253 intact lipid species and 18 fatty acids across 4 lipid classes were recognized in plasma samples, while FA C20:3 was confirmed as the fatty acyl chain belonging to phosphatidylinositol, PI 38:3, which was found to be down-regulated in fibroblast samples of Parkinson&rsquo, s disease patients.

An extensive characterization and quantification of intact phospholipids (PLs) in strawberry (Fragaria × ananassa cv San Andreas) seed and pulp was carried out by hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization (ESI) coupled to either Fourier-transform (FT) orbital-trap or linear ion-trap tandem mass spectrometry (LIT-MS/MS). More than 150 intact polar lipids including phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylglycerols (PGs), phosphatidic acids (PAs), phosphatidylinositols (PIs), lysophosphatidylcholines (LPCs), and lysophosphatidylethanolamines (LPEs) were identified in negative ESI mode. PC 18:2/18:2 and 18:2/18:3 were found to be the major components of strawberry lipid extracts at concentrations of 230 ± 36 and 189 ± 32 μg/g, respectively, in seeds and at concentrations of 330 ± 50 and 140 ± 22 μg/g, respectively, in pulp. The lipidic extracts of both strawberry seeds and pulp exhibited the dominance of LPC 16:0/0:0 at a content of 132 ± 19 and 114 ± 16 μg/g, respectively, and LPC 0:0/18:2 at 236 ± 20 and 150 ± 20 μg/g, respectively. The other most abundant species of strawberry seeds and pulp were PE 18:2/18:2, 40 ± 9 and 190 ± 40 μg/g, followed by PI 16:0/18:2, 51 ± 15 and 24 ± 8 μg/g, respectively, while PG, PA, and LPE show comparable abundance below 10 μg/g. The most recurrent fatty acyl substituents of PLs were C18:3 (α-linolenic acid), C18:2 (linoleic acid), C18:1 (oleic acid), C18:0 (stearic acid), C16:0 (palmitic acid), and relatively high contents of a shorter chain such as C14:0 (myristic acid).

The issue of deliberate addition of antigenic proteins to foodstuffs for ameliorating bulk properties or the unintentional cross-contamination poses potentially life-threatening health problems to susceptible subjects. Even the intake of food products declaring the absence of allergens on their labels could lead to severe risks for sensitive consumers due to the presence of the so-called “hidden allergens”. Thus, the quantification of low-abundant proteins as putative allergens has become mandatory. Herein, we present a sensitive and selective analytical method based on reversed-phase liquid chromatography coupled to electrospray ionization and hybrid orbitrap high-resolution mass spectrometry (RPLC-ESI-HRMS) and tandem MS, identifying, and quantifying allergenic milk proteins in complex meat-based foodstuffs from direct measurement of tryptic peptides. Two signature peptides of α-S1-casein and β-lactoglobulin, i.e., FFVAPFPEVFGK (m/z 692.8682+) and TPEVDDEALEK (m/z 623.2952+), respectively, were chosen to search for hidden allergens in meat-based samples such as cooked meat, sausages, and sterilised pâte. The marker peptides were identified and were exploited for method validation including recovery, matrix effect, precision, linearity, method variation, limit of detection, and limit of quantification. The undeclared occurrence of milk allergens as total milk protein content (TCMP) was verified in commercial meat products; beef and pork pâte were meat-based products which require a major alert because up to 22 μgTCMP/g of matrix i.e. more than 10 times the action level was determined.

Pt(II)-based derivatives bearing a cyanocobalamin (CNCbl) unit were synthesized in aqueous solutions, and the reaction mixtures were examined by reversed-phase liquid chromatography with electrospray ionization and linear ion trap mass spectrometry (MS). Isotopic pattern analysis, multistage mass-spectra (MS/MS and MS3) interpretation, and differential isotopic labeling were used to establish the chemical composition and to suggest the chemical structures of reaction products. When cisplatin (cis-[PtCl2(NH3)2]) was used as a Pt(II) drug derivative, a coordination bond between diamminemonochloroplatinum(II) and the cyano group of CNCbl, in turn linked covalently to the vitamin Co(III) ion, occurred. The resulting conjugate with a CoIII–CN–PtII bridge was MS detected as a doubly positive charged ion with the prevailing isotopologue at m/z 810.26 (empirical formula [C63H95ClCoIIIN16O14PPt]2+). Likewise, a peak signal centered at m/z 811.26 was observed when 15N-labeled cisplatin cis-[PtCl2(15NH3)2] was used ...

Gold nanomaterials hold great potential for biomedical applications. While this field is evolving rapidly, little attention has been paid to precise nanoparticle design and functionalization. Here, we show that when using proteins as targeting moieties, it is fundamental to immobilize them directionally to preserve their biological activity. Using full-length leptin as a case study, we have developed two alternative conjugation strategies for protein immobilization based on either a site-selective or a nonselective derivatization approach. We show that only nanoparticles with leptin immobilized site-selectively fully retain the ability to interact with the cognate leptin receptor. These results demonstrate the importance of a specified molecular design when preparing nanoparticles labeled with proteins.

cis-Diamminedichloroplatinum(II), also known as cisplatin, is a widely used chemotherapeutic agent to treat several malignant tumours, but unfortunately it causes serious side effects, especially nausea, vomiting and nephrotoxicity. Enhancing the selectivity to cancer cells using other compounds combined with cisplatin may overcome these issues. Here, cisplatin and oxaliplatin derivatives bearing a cyanocobalamin (CNCbl) unit were prepared and investigated by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). A 4-chloro-α-cyanocinnamic acid (ClCCA) matrix was very effective to explore the formation of CNCbl–Pt(II) conjugates. In the case of cisplatin, the conjugate is formed by the elimination of one easily leaving anionic ligand (i.e., Cl−) from the complex, whereas the cyano group (CN) that is axially coordinated to Co(III) in CNCbl (M) becomes bound to the Pt(II) of monochloro cisplatin, thus yielding a heterobimetallic derivative with the most intense peak of the ion cluster at m/z 1619.55 ([M + cis-PtII (NH3)2Cl]+) and an empirical formula of [C63H94ClCoIIIN16O14PPtII]+. Its identity was revealed by tandem MS (CID ToF/ToF) and the subsequent recognition of the most intense product ions. Likewise, the chelating (1R,2R)-1,2-diaminocyclohexane (R,R-DACH) non-leaving group ligand of oxaliplatin, in the form of cis-sulfoaquo R,R-DACH–Pt(II), produces two different conjugates with CNCbl in MALDI-MS ([M + PtII(R,R-DACH)]+) at m/z 1663.64 and 1664.63, both singly-positively charged, with suggested compositions of [C69H101CoIIIN16O14PPtII]+ and [C69H102CoIIIN16O14PPtII]+˙, respectively. By properly relying on tandem MS data, it has been possible to propose a detailed description of these two CNCbl–Pt(II) drug conjugates. This investigation sets the stage for future MALDI MS studies on platinum anticancer drugs.

Early diagnosis of neural changes causing cerebral impairment is critical for proposing preventive therapies for Parkinson&rsquo, s disease (PD). Biomarkers currently available cannot be informative of PD onset since they are characterized by analysing post-mortem tissues from patients with severe degeneration of the substantia nigra. Skin fibroblasts (SF) are now recognized as a useful model of primary human cells, capable of reflecting the chronological and biological aging of the subjects. Here a lipidomic study of easily accessible primary SF is presented, based on hydrophilic interaction liquid chromatography coupled to electrospray ionization and mass spectrometry (HILIC/ESI-MS). Phospholipids (PL) from dermal fibroblasts of five PD patients with different parkin mutations and healthy control SF were characterized by single and tandem MS measurements using a hybrid quadrupole-Orbitrap and a linear ion trap mass analysers. The proposed approach enabled the identification of more than 360 PL. Univariate statistical analyses highlight abnormality of PL metabolism in the PD group, suggesting down- or up-regulation of certain species according to the extent of disease progression. These findings, although preliminary, suggest that the phospholipidome of human SF represents a source of potential biomarkers for the early diagnosis of PD. The dysregulation of ethanolamine plasmalogens in the circulatory system, especially those containing polyunsaturated fatty acids (PUFA), might be likely associated with neurodegeneration.

Bacteriochlorophyll a (BChl a), a photosynthetic pigment performing the same functions of chlorophylls in plants, features a bacteriochlorin macrocycle ring (18 π electrons) with two reduced pyrrole rings along with a hydrophobic terpenoid side chain (i.e., the phytol residue). Chlorophylls analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is not so straightforward since pheophytinization (i.e., release of the central metal ion) and cleavage of the phytol–ester linkage are invariably observed by employing protonating matrices such as 2,5-dihydroxybenzoic acid, sinapinic acid, and α-cyano-4-hydroxycinnamic acid. Using BChl a from Rhodobacter sphaeroides R26 strain as a model system, different electron-transfer (ET) secondary reaction matrices, leading to the formation of almost stable radical ions in both positive ([M]+•) and negative ([M]−•) ionization modes at m/z 910.55, were evaluated. Compared with ET matrices such as trans-2-[3-(4-t-butyl-phenyl)-2-methyl-2-propenylidene]malononitrile (DCTB), 2,2':5',2''-terthiophene (TER), anthracene (ANT), and 9,10-diphenylanthracene (DP-ANT), 1,5-diaminonaphthalene (DAN) was found to provide the highest ionization yield with a negligible fragmentation. DAN also displayed excellent ionization properties for two metal ion-substituted bacteriochlorophylls, (i.e., Zn- and Cu-BChl a at m/z 950.49 and 949.49), respectively. MALDI MS/MS of both radical charged molecular species provide complementary information, thus making analyte identification more straightforward.

4-Chloro-α-cyanocinnamic acid (ClCCA) is a very useful matrix able to give the protonated adduct [M+H](+) of intact cyanocobalamin (CNCbl) as the base peak (m/z 1355.58) in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The only fragment observed is [M-CN + H](+•) formed through the facile (•) CN neutral loss reflecting the fairly low Co-C bond energy. All other investigated proton transfer matrices, including α-cyano-4-hydroxycinnamic acid, para-nitroaniline and 2,5-dihydroxybenzoic acid, give rise to a complete decyanation of CNCbl with concomitant formation of [M-CN + H](+•) , [M-CN + Na](+•) and [M-CN + K](+•) adducts at m/z 1329.57, 1351.55 and 1367.51, respectively. Depending on the matrix used, a variable degree of fragmentation involving the α-side axial ligand was observed. A plausible explanation of the specific behaviour of 4-chloro-α-cyanocinnamic acid as a soft matrix is discussed. Tandem mass spectra of both [M + H](+) and [M-CN + H](+•) ions were obtained and product ions successfully assigned. The possibility of detecting the protonated adduct of intact CNCbl was exploited in foodstuff samples such as cow milk and hen egg yolk by MALDI tandem MS upon sample extraction. We believe that our data provide strong basis for the application of MALDI tandem MS in the qualitative analysis of natural CNCbl, including fish, liver and meat samples. Copyright © 2016 John Wiley & Sons, Ltd.

Hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization (ESI) coupled to either Fourier-transform (FT) orbital-trap or linear ion-trap tandem mass spectrometry (LIT-MS/MS) was used to characterize the phospholipidome of yellow lupin (Lupinus luteus) seeds. Phosphatidylcholines (PC) were the most abundant species (41 &plusmn, 6%), which were followed by lyso-forms LPC (30 &plusmn, 11%), phosphatidylethanolamines (PE, 13 &plusmn, 4%), phosphatidylglycerols (PG, 5.1 &plusmn, 1.7%), phosphatidic acids (PA, 4.9 &plusmn, 1.8%), phosphatidylinositols (PI, 4.7 &plusmn, 1.1%), and LPE (1.2 &plusmn, 0.5%). The occurrence of both isomeric forms of several LPC and LPE was inferred by a well-defined fragmentation pattern observed in negative ion mode. An unprecedented characterization of more than 200 polar lipids including 52 PC, 42 PE, 42 PA, 35 PG, 16 LPC, 13 LPE, and 10 PI, is reported. The most abundant fatty acids (FA) as esterified acyl chains in PL were 18:1 (oleic), 18:2 (linoleic), 16:0 (palmitic), and 18:3 (linolenic) with relatively high contents of long fatty acyl chains such as 22:0 (behenic), 24:0 (lignoceric), 20:1 (gondoic), and 22:1 (erucic). Their occurrence was confirmed by reversed-phase (RP) LC-ESI-FTMS analysis of a chemically hydrolyzed sample extract in acid conditions at 100 &deg, C for 45 min.

Chlorophylls (Chls) are important pigments responsible for the characteristic green color of chloroplasts in algae and plants. In this study, 1,5-diaminonaphthalene (DAN) was introduced as an electron transfer secondary reaction matrix for the identification of intact chlorophylls and their derivatives, by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). DAN was proved to drastically outperform conventional matrices such as α-cyano-4-hydroxycinnnamic acid, dithranol, antracene, and even terthiophene, since loss of the metal ion and fragmentation of the phytol-ester linkage are negligible. Absence of significant fragmentation of radical cations of Chls a and b at m/z 892.529 and 906.513, respectively, makes MALDI MS capable of following natural degradation of intact porphyrin-based pigments whose initial steps are just represented by demetalation and dephytylation. Chl by-products, such as pyropheophytins, have been identified in dried tea leaves showing the potential of MALDI MS to follow chlorophyll biotransformation occurring in processed foodstuffs. Finally, preliminary results show the potential of MALDI MS to detect illegal vegetable oil re-greening practices.

Skin fibroblasts are recognized as a valuable model of primary human cells able of mirroring the chronological and biological aging. Here, a lipidomic study of glycosphingolipids (GSL) occurring in the easily accessible human dermal fibroblasts (HDF) is presented. Reversed-phase liquid chromatography with negative electrospray ionization (RPLC-ESI) coupled to either orbitrap or linear ion-trap multiple-stage mass spectrometry was applied to characterize GSL in commercially adult and neonatal primary human fibroblast cells and in skin samples taken from an adult volunteer. Collision-induced dissociation in negative ion mode allowed us to get information on the monosaccharide number and ceramide composition, whereas tandem mass spectra on the ceramide anion was useful to identify the sphingoid base. Nearly sixty endogenous GSL species were successfully recognized, namely 33 hexosyl-ceramides (i.e., HexCer, Hex2Cer and Hex3Cer) and 24 gangliosides as monosialic acid GM1, GM2 and GM3, along with 5 globosides Gb4. An average content of GSLs was attained and the most representative GSL in skin fibroblasts were Hex3Cer, also known as Gb3Cer, followed by Gb4, HexCer and Hex2Cer , while gangliosides were barely quantifiable. The most abundant GSLs in the examined cell lines share the same ceramide base (i.e. d18:1) and the relative content was d18:1/24:1 > d18:1/24:0 > d18:1/16:0 > d18:1/22:0.