Unambiguous regiochemical assignment of sulfoquinovosyl mono- and diacylglycerols in parsley and spinach leaves by liquid chromatography/electrospray ionization sequential mass spectrometry assisted by regioselective enzymatic hydrolysis.

RATIONALE: Sulfoquinovosylmonoglycerides (SQMG) and sulfoquinovosyldiglycerides (SQDG) in the lipid extracts of parsley (Petroselinum crispum) and spinach (Spinacia oleracea) leaves were investigated. The aim of this work was to assess and establish the chemical characterization of fatty acyl chains in sulfolipids (SQMG and SQDG) and their regiochemistry. METHODS: A key component of this approach is a combination of hydrolysis reactions catalyzed by Lecitase® Ultra, which is a sn1 -regioselective hydrolase enzyme, and reversed-phase liquid chromatography with electrospray ionization and sequential mass spectrometry (RPLC/ESI-MS) by collision-induced dissociation (CID)-MSn (n = 2, 3). RESULTS: The occurrence of SQMG bearing 16:0 or 18:3 acyl chains was established for the first time. A regiochemistry-dependent fragmentation pattern of SQMG was attained whereby the sulfoquinovosyl anion ([C6 H11 O8 S]- at m/z 243.0) provides a diagnostic product ion. Regioselective enzymatic treatment also provided a posteriori confirmation of a widely accepted fragmentation rule for SQDG. The sulfoquinovosyl anion was found to play a role also in the fragmentation pattern of SQDG, whose regiochemical assignment could be ultimately confirmed by MS3 experiments. CONCLUSIONS: The predominant sulfolipid in leaf extracts of raw parsley (Petroselinum crispum) and spinach (Spinacia oleracea) was identified as SQDG 18:3/16:0, along with SQMG 18:3/0:0 and SQMG 16:0/0:0. The present CID-MS-based method can be considered a successful approach to validate the regiochemical characterization of sulfolipids paving the way for their unambiguous characterization.

Unveiling the compositional variety of cardiolipins in Rhodobacter sphaeroides by liquid chromatography with electrospray ionization and multistage collision-induced dissociation mass spectrometry.

Cardiolipins (CL) contained in the lipid extracts of the photosynthetic bacterium Rhodobacter sphaeroides (strain R26) were systematically characterized by reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry, performed in single (MS), tandem (MS/MS) and sequential (MS3) modes using a linear ion trap mass spectrometer. The total number of carbon atoms and C=C bonds of each CL and, subsequently, those related to each of the constituting phosphatidic acid (PA) units, along with the location of the latter on the central glycerol backbone, were inferred from MS and MS/MS data, respectively. Moreover, the composition and location of both acyl chains on the glycerol backbone of each PA unit was obtained by MS3 measurements, an approach used for the first time for the structural elucidation of CL in R. sphaeroides. As a result, an unprecedented profile of CL in this bacterium was obtained, with 27 main species characterized, many of which are represented by compositional or regiochemical isomers. Interestingly, such a variability is generated from a limited number of different acyl chains, either saturated (i.e. 12:0, 16:0, 17:0, 18:0, 19:0) or mono-unsaturated (16:1, 18:1). The absence of polyunsaturated chains, more susceptible to oxidation damage, appeared to be indirectly related to the lack of carotenoids potentially acting as antioxidant agents, a specific feature of R. sphaeroides R26. The occurrence of odd-numbered acyl chains was ascribed to the need to guarantee membrane fluidity, through a less compact packing of CL, thus compensating for the lack of CL bearing polyunsaturated side chains. Graphical abstract Representation of MS signals due to carboxylate anions that would be detected, as separate couples, in the fragmentation spectra of the anions resulting from the two phosphatidic acid units included in a cardiolipin molecule bearing four different acyl chains.

Identification of isobaric lyso-phosphatidylcholines in lipid extracts of gilthead sea bream (Sparus aurata) fillets by hydrophilic interaction liquid chromatography coupled to high-resolution Fourier-transform mass spectrometry.

The numerous and varied biological roles of phosphatidylcholines (PC) and lysophosphatidylcholines (LPC) have fueled a great demand for technologies that enable rapid, in-depth structural examination of these lipids in foodstuffs. Here, we describe the capabilities of a newly configured combination of high-efficiency liquid chromatography and high-resolution/accuracy Fourier-transform mass spectrometry with electrospray ionization (LC-ESI-FTMS), designed for lipidomics applications that require the identification of PC in their lyso forms. The devised strategy, involving a separation by hydrophilic interaction liquid chromatography (HILIC) on spherical, fused-core ultrapure silica particles (2.7 µm) of a narrow-bore column (2.1 mm i.d.), enabled the identification of as many as 71 LPC species in the lipid extracts of gilthead sea bream (Sparus aurata) fillets. In this way, LPC as proton (43) and sodium (28) adducts, i.e., [M + H](+) and [M + Na](+) ions (with M representing the zwitterionic form), were identified. In several cases, the extremely high (sub-ppm) mass accuracy and the high chromatographic efficiency available with the adopted instrumentation enabled the distinction of isobaric and closely eluting LPC species. Informative tandem mass spectra, based on high-energy collision induced dissociation (HCD), were also obtained, thus distinguishing regioisomeric LPC species (i.e., LPC differing only for the location of the residual side chain on the glycerol backbone) and between proton and sodium adducts. Graphical Abstract Extracted Ion Current chromatogram (XIC) obtained for the m/z value 568.339, showing the presence of two regioisomeric Lysophosphatidylcholines. The corresponding high collisional energy tandem MS spectra, obtained using a HCD cell, are shown as insets.

Hydrophilic interaction and reversed phase mixed-mode liquid chromatography coupled to high resolution tandem mass spectrometry for polar lipids analysis.

A hydrophilic interaction liquid chromatography (HILIC) fused-core column (150×2.1mm ID, 2.7µm particle size) and a short reversed-phase liquid chromatography (RPLC) column (20mm×2.1mm ID, 1.9µm) were serially coupled to perform mixed-mode chromatography (MMC) on complex mixtures of phospholipids (PL). Mobile phase composition and gradient elution program were, preliminarily, optimized using a mixture of phosphatidylcholines (PC), phosphatidylethanolamines (PE), their corresponding lyso-forms (LPC and LPE), and sphingomyelins (SM). Thus a mixture of PC extracted from soybean was characterized by MMC coupled to electrospray ionization (ESI) high-resolution Fourier-transform mass spectrometry (FTMS) using an orbital trap analyzer. Several previously undiscovered PC, including positional isomers (i.e. 16:0/19:1 and 19:1/16:0) of PC 35:1 and skeletal isomers (i.e. 18:1/18:2 and 18:0/18:3) of PC 36:3 were identified. Therefore, high-resolution MS/MS spectra unveiled the occurrence of isomers for several overall side chain compositions. The proposed MMC-ESI-FTMS/MS approach revealed an unprecedented capability in disclosing complexity of an actual lipid extract, thus representing a very promising approach to lipidomics.

Profiling of ornithine lipids in bacterial extracts of Rhodobacter sphaeroides by reversed-phase liquid chromatography with electrospray ionization and multistage mass spectrometry (RPLC-ESI-MS(n)).

Ornithine lipids (OLs), a sub-group of the large (and of emerging interest) family of lipoamino acids of bacterial origin, contain a 3-hydroxy fatty acyl chain linked via an amide bond to the α-amino group of ornithine and via an ester bond to a second fatty acyl chain. OLs in extracts of Rhodobacter sphaeroides (R. sphaeroides) were investigated by high-performance reversed phase liquid chromatography (RPLC) with electrospray ionization mass spectrometry (ESI-MS) in negative ion mode using a linear ion trap (LIT). The presence of OLs bearing both saturated (i.e, 16:0, 17:0, 18:0, 19:0 and 20:0) and unsaturated chains (i.e., 18:1, 19:1, 19:2 and 20:1) was ascertained and their identification, even for isomeric, low abundance and partially co-eluting species, was achieved by low-energy collision induced dissociation (CID) multistage mass spectrometry (MS(n), n = 2-4). OLs signatures found in two R. sphaeroides strains, i.e., wild type 2.4.1 and mutant R26, were examined and up to 16 and 17 different OL species were successfully identified, respectively. OLs in both bacterial strains were characterized by several combinations of fatty chains on ester-linked and amide-linked 3-OH fatty acids. Multistage MS spectra of monoenoic amide-linked 3-OH acyl chains, allowed the identification of positional isomer of OL containing 18:1 (i.e. 9-octadecenoic) and 20:1 (i.e. 11-eicosenoic) fatty acids. The most abundant OL ([M-H](-) at m/z 717.5) in R. sphaeroides R26 was identified as OL 3-OH 20:1/19:1 (i.e., 3-OH-eicosenoic acid amide-linked to ornithine and esterified to a nonadecenoic chain containing a cyclopropane ring). An unusual OL (m/z 689.5 for the [M-H](-) ion), most likely containing a cyclopropene ester-linked acyl chain (i.e., OL 3-OH 18:0/19:2), was retrieved only in the carotenoidless mutant strain R26. Based on the biosynthetic pathways already known for cyclopropa(e)ne ring-including acyl chains, a plausible explanation was invoked for the enzymatic generation of this ester-linked chain in R. sphaeroides.

Ancient pottery from archaeological sites in Southern Italy: first evidence of red grape products markers.

The chemical analysis of tartaric acid (TA) and syringic acid (SA), as grape product markers in ancient ceramic vessels from the sites of Manduria and Torre di Satriano (southern Italy), was successfully performed. Firstly, the fragmentation behaviour of TA and SA as deprotonated molecules, [M-H](-), obtained by collision-induced dissociation, was investigated. Then, reversed-phase liquid chromatography (RPLC) with electrospray ionization (ESI) in negative ion mode, using a quadrupole linear ion trap in multiple reaction monitoring (MRM), was employed. A binary mobile phase composed of water-acetonitrile with 0.1% (v/v) acetic acid enabled the optimum ESI efficiency of SA, greatly improving its identification when it occurs in trace amounts. Chemical analysis of ancient pottery fragments is a valid method for establishing the existence of preserved organic residues, which is valuable new evidence for the culture and customs of ancient populations, in this case those of southern Italy. The proposed RPLC-ESI-MRM method allowed a systematic investigation of ceramic fragments of both archaeological sites, thus providing positive evidence for the presence of TA and SA as grape product markers in storage vessels dating back to the ninth to third centuries BC.

Occurrence of oleic and 18:1 methyl-branched acyl chains in lipids of Rhodobacter sphaeroides 2.4.1.

The fatty acids (FAs) composition of lipids extracted from Rhodobacter sphaeroides 2.4.1 was investigated by gas chromatography-mass spectrometry (GC-MS) analysis of the corresponding FA methyl esters (FAMEs), obtained through trans-esterification of the original lipid species. A GC stationary phase based on a highly polar ionic liquid (IL) was selected, aimed to enhance the separation of isomeric FAMEs with particular emphasis on positional and geometrical isomers of monounsaturated 16:1 and 18:1 fatty acyl chains. The occurrence of 18:1 cis-Δ(9) (oleic) acid, a positional isomer of the well-known and most predominant 18:1 cis-Δ(11) (cis-vaccenic) acid, has been demonstrated here for the first time. Furthermore a methyl branched 18:1 FA was also identified and its structure tentatively assigned as 11-methyl-Δ(12)-octadecenoic acid (most likely as trans isomer). The unprecedented observation about 18:1 cis-Δ(9) FA occurrence in R. sphaeroides 2.4.1 is, even indirectly, supported by a biosynthetic pathway postulated with the aid of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The concurrent presence of 16:1 cis-Δ(7) and 18:1 cis-Δ(9) FAs suggested the existence of parallel and/or complementary processes to those invoked for the formation of most common 16:1 cis-Δ(9) and 18:1 cis-Δ(11) FAs. A further route was hypothesized for the trans FAs biosynthesis in wild-type cells of R. sphaeroides.