Using the Synergy between HPLC-MS and MALDI-MS Imaging to Explore the Lipidomics of Clear Cell Renal Cell Carcinoma.

Lipid imaging mass spectrometry (LIMS) has been tested in several pathological contexts, demonstrating its ability to segregate and isolate lipid signatures in complex tissues, thanks to the technique's spatial resolution. However, it cannot yet compete with the superior identification power of high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS), and therefore, very often, the latter is used to refine the assignment of the species detected by LIMS. Also, it is not clear if the differences in sensitivity and spatial resolution between the two techniques lead to a similar panel of biomarkers for a given disease. Here, we explore the capabilities of LIMS and HPLC-MS to produce a panel of lipid biomarkers to screen nephrectomy samples from 40 clear cell renal cell carcinoma patients. The same set of samples was explored by both techniques, and despite the important differences between them in terms of the number of detected and identified species (148 by LIMS and 344 by HPLC-MS in negative-ion mode) and the presence/absence of image capabilities, similar conclusions were reached: using the lipid fingerprint, it is possible to set up classifiers that correctly identify the samples as either healthy or tumor samples. The spatial resolution of LIMS enables extraction of additional information, such as the existence of necrotic areas or the existence of different tumor cell populations, but such information does not seem determinant for the correct classification of the samples, or it may be somehow compensated by the higher analytical power of HPLC-MS. Similar conclusions were reached with two very different techniques, validating their use for the discovery of lipid biomarkers.

A UHPLC-Mass Spectrometry View of Human Melanocytic Cells Uncovers Potential Lipid Biomarkers of Melanoma.

Melanoma is the deadliest form of skin cancer due to its ability to colonize distant sites and initiate metastasis. Although these processes largely depend on the lipid-based cell membrane scaffold, our understanding of the melanoma lipid phenotype lags behind most other aspects of this tumor cell. Here, we examined a panel of normal human epidermal and nevus melanocytes and primary and metastatic melanoma cell lines to determine whether distinctive cell-intrinsic lipidomes can discern non-neoplastic from neoplastic melanocytes and define their metastatic potential. Lipidome profiles were obtained by UHPLC-ESI mass-spectrometry, and differences in the signatures were analyzed by multivariate statistical analyses. Significant and highly specific changes in more than 30 lipid species were annotated in the initiation of melanoma, whereas less numerous changes were associated with melanoma progression and the non-malignant transformation of nevus melanocytes. Notably, the "malignancy lipid signature" features marked drops in pivotal membrane lipids, like sphingomyelins, and aberrant elevation of ether-type lipids and phosphatidylglycerol and phosphatidylinositol variants, suggesting a previously undefined remodeling of sphingolipid and glycerophospholipid metabolism. Besides broadening the molecular definition of this neoplasm, the different lipid profiles identified may help improve the clinical diagnosis/prognosis and facilitate therapeutic interventions for cutaneous melanoma.

A simple and reproducible method for quantification of human tear lipids with ultrahigh-performance liquid chromatography-mass spectrometry.

Purpose: The purpose was to select a simple and reproducible method for lipid measurements of human tears with ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS). Two sample preparation procedures were evaluated and compared: the Bligh and Dyer (BD) liquid-liquid extraction method with chloroform and methanol and protein precipitation with isopropanol (IPA). Methods: Reproducibility and recovery efficiencies of 20 non-endogenous internal lipid standards were tested in 10-µl tear samples from healthy subjects. The lipid coverage and the simplicity of execution were also assessed. Lipid profiles of the tear extracts were acquired with UHPLC-MS, uhpland the lipids were identified using SimLipid software. Results: Both methods were robust producing good lipid coverage and reproducibility and high recovery efficiencies. The two protocols identified a 69-feature tear lipidome that covered 11 lipid classes from six different lipid categories. The main differences in recovery were due to the intrinsic lipid selectivity of each solvent. Although both methods were similarly efficient in recovering O-acyl-ω-hydroxy fatty acid (OAHFAs) and non-polar lipids, polar lipids were more efficiently recovered with IPA precipitation, which, in turn, exhibited higher reproducibility. In addition, IPA precipitation is automatable and simpler than the BD approach. Conclusions: IPA precipitation is an excellent procedure for extracting lipids from small tear volumes for quantitative large-scale, untargeted lipid profiling, which may be useful for identifying lipid biomarkers in tears from patients with different ocular surface pathologies, allowing personalized therapies to be designed.

Untargeted Metabolomic Liquid Chromatography High-Resolution Mass Spectrometry Fingerprinting of Apple Cultivars for the Identification of Biomarkers Related to Resistance to Rosy Apple Aphid.

Liquid chromatography high-resolution mass spectrometry fingerprinting together with pattern recognition techniques was used to determine the metabolites involved in the susceptibility of apple cultivars to rosy apple aphid (RAA). Preprocessing of ultra-high-performance liquid chromatography coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry raw data of resistant and susceptible apple cultivars was carried out with XCMS and CAMERA packages. Univariate statistical tools and multivariate data analysis highlighted significant different profiles of the apple metabolomes according to their tolerance to RAA. Optimized and cross-validated Partial least squares discriminant analysis and orthogonal projections to latent structures discriminant analysis models confirmed trans-4-caffeoylquinic acid and 4-p-coumaroylquinic acid as biomarkers for the identification of resistant and susceptible apple cultivars to RAA and disclosed that only hydroxycinnamic acids are involved in the disease susceptibility of cultivars. In this sense, the final steps of the biosynthesis of caffeoylquinic acid (CQA) and p-coumaroylquinic acid (p-CoQA) become decisive because the isomerization of 5-CQA to 4-CQA is favored in resistant cultivars, whereas the isomerization of 5-p-CoQA to 4-p-CoQA is favored in susceptible cultivars.

Improving spatial resolution of a LTQ Orbitrap MALDI source.

Here we present a simple and cost effective procedure to improve the spatial resolution of the commercial MALDI source of a LTQ Orbitrap. Based in spatial filtering techniques, we demonstrate that, with minimal modifications of the original set up, the system resolution can be pushed forward to <10 µm. The improved system performance is demonstrated by means of MALDI imaging of human colon biopsies.

Cerebrospinal Fluid 7-Ketocholesterol Level is Associated with Amyloid-ß42 and White Matter Microstructure in Cognitively Healthy Adults.

BACKGROUND: Abnormal cholesterol metabolism changes the neuronal membrane and may promote amyloidogenesis. Oxysterols in cerebrospinal fluid (CSF) are related to Alzheimer's disease (AD) biomarkers in mild cognitive impairment and dementia. Cholesterol turnover is important for axonal and white matter (WM) microstructure maintenance. OBJECTIVE: We aim to demonstrate that the association of oxysterols, AD biomarkers, and WM microstructure occurs early in asymptomatic individuals. METHODS: We studied the association of inter-individual variability of CSF 24-hydroxycholesterol (24-OHC), 27-hydroxycholesterol (27-OHC), 7-ketocholesterol (7-KC), 7ß-hydroxycholesterol (7ß-OHC), amyloid-ß42 (Aß42), total-tau (t-tau), phosphorylated-tau (p-tau), neurofilament (NfL), and WM microstructure using diffusion tensor imaging, generalized linear models and moderation/mediation analyses in 153 healthy adults. RESULTS: Higher 7-KC levels were related to lower Aß42, indicative of greater AD pathology (p = 0.041) .  Higher 7-KC levels were related to lower fractional anisotropy (FA) and higher mean (MD), axial (AxD), and radial (RD) diffusivity. 7-KC modulated the association between AxD and NfL in the corpus callosum splenium (B = 39.39, p = 0.017), genu (B = 68.64, p = 0.000), and fornix (B = 10.97, p = 0.000). Lower Aß42 levels were associated to lower FA and higher MD, AxD, and RD in the fornix, corpus callosum, inferior longitudinal fasciculus, and hippocampus. The association between AxD and Aß42 was moderated by 7K-C (p = 0.048). CONCLUSION: This study adds clinical evidence to support the role of 7K-C on axonal integrity and the involvement of cholesterol metabolism in the Aß42 generation process.

A fragmentation study of dihydroquercetin using triple quadrupole mass spectrometry and its application for identification of dihydroflavonols in Citrus juices.

A mass spectrometric method using electrospray ionization with triple quadrupole and quadrupole time-of-flight hybrid (Q-Tof) mass spectrometry has been applied to the structural characterization of dihydroflavonols. This family of compounds has been studied by liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the first time in this work. A comprehensive study of the product ion MS spectra of the [M+H](+) ion of a commercially available standard has been performed. The most useful fragmentations in terms of structural identification are those that involve cleavage of the C-ring, resulting in diagnostic ions of dihydroflavonol family: (1,3)A(0) (+), (1,2)B(0) (+), (1,2)B(0) (+)-CO, (0,2)A(0) (+), (0,2)A(0) (+)-H(2)O, (0,2)A(0) (+)-CO, and (0,2)A(0) (+)-H(2)O-CO, that allow the characterization of the substituents in the A- and B-rings. In addition to those ions, other product ions due to losses of H(2)O and CO molecules from the Y(0) (+) ion were observed. Their fragmentation mechanisms and ion structures have been proposed. The established fragmentation patterns have been used to successfully identity three dihydroflavonols found in tangerine juices for the first time.

Lipidomic data uncover extensive heterogeneity in phosphatidylcholine structural variants in HepG2 cells.

The data contain information related to the research article entitled "Profiling of promoter occupancy by the SND1 transcriptional coactivator identifies downstream glycerolipid metabolic genes involved in TNFα response in human hepatoma cells" (DOI: 10.1093/nar/gkv858). In the article alluded to, we reported that tumor necrosis factor alpha (TNFα) increases notably the cellular content of the major glycerolipid phosphatidylcholine (PC). Here, accompanying lipidomic data determine the PC structural variants that have been identified in human hepatoma HepG2 cells and those whose relative abundance is modified by TNFα. We used ultrahigh performance liquid chromatography (UHPLC) coupled to electrospray ionization (ESI) tandem mass spectrometry (MS/MS)-based lipidomic profiling to analyze lipid extracts of control and TNFα-treated HepG2 cells. The identity of PC individual species was elucidated using the values of the retention time and molecular weight in addition to the fragmentation patterns. MS data were then processed and analyzed for the characterization of statistically significant differences in detected structural variants. We have annotated the dataset of PC species that characterize HepG2 cells' phenotype, both under normal and pro-inflammatory conditions.

Isopropanol extraction for cerebrospinal fluid lipidomic profiling analysis.

The cerebrospinal fluid (CSF) lipidome is attracting increasing attention due to the importance of lipids in brain molecular signaling and their involvement in several neurological diseases. Different solvent systems have been used for the extraction of multiple lipid classes from CSF but no comparative study of the effectiveness of these protocols has been carried out. To optimize CSF lipid extraction for lipidomic measurements by untargeted ultra-high performance liquid chromatography - mass spectrometry, we evaluate and compare two sample preparation protocols, one involving protein precipitation with isopropanol (IPA) and other consisting of a liquid-liquid extraction with chloroform-methanol. For that purpose, human CSF from neurologically healthy and normolipidemic volunteers was used. The criteria established to compare these two methods were based on four critical aspects of sample preparation: simplicity, lipid coverage, reproducibility and recovery efficiencies. We found that both methods were highly reproducible techniques (>75% of the lipids with coefficient of variation (CV) <30%). In terms of recovery, the single-step IPA procedure yielded better values for most of the lipid classes and it was less toxic and simpler than the liquid-liquid extraction method. In relation to lipid coverage, variation in selectivity was observed between methods, providing evidence that IPA was more selective for polar lipids. Overall, IPA precipitation provides excellent results in terms of simplicity of execution, lipid coverage, reproducibility and recovery. We conclude that it is a choice procedure for large-scale, untargeted lipid profiling using UHPLC-MS in CSF analysis.

Detection of non-coloured anthocyanin-flavanol derivatives in Rioja aged red wines by liquid chromatography-mass spectrometry.

Anthocyanins, responsible for wine colour, are involved in many reactions during wine ageing. Anthocyanin-flavanol associations give rise to derivatives in flavylium form that provide blue hues, but also derivatives that do not directly influence wine colour. These colourless derivatives remain mostly unknown but their roles during wine ageing are important for controlling wine quality. Colourless anthocyanin-flavanol derivatives formed during wine ageing have been studied in three aged red wines from Rioja using a combined method with Column Chromatography (CC) and High Performance Liquid Chromatography with Diode Array and Mass Spectrometric detections (HPLC-DADMS). Twenty-six compounds have been detected: 17 dimers with the anthocyanin in flavene form with possible anthocyanin-flavanol (type 1) and flavanol-anthocyanin (type 2) structures, and 9 with an A-type bicyclic anthocyanin-flavanol structure (type 3). Although some of malvidin derivatives have been previously reported, this is the first time that these derivatives (including different isomers) have also been detected for delphinidin, petunidin and peonidin.

Analysis of the lipidome of xenografts using MALDI-IMS and UHPLC-ESI-QTOF.

Human tumor xenografts in immunodeficient mice are a very popular model to study the development of cancer and to test new drug candidates. Among the parameters analyzed are the variations in the lipid composition, as they are good indicators of changes in the cellular metabolism. Here, we present a study on the distribution of lipids in xenografts of NCI-H1975 human lung cancer cells, using MALDI imaging mass spectrometry and UHPLC-ESI-QTOF. The identification of lipids directly from the tissue by MALDI was aided by the comparison with identification using ESI ionization in lipid extracts from the same xenografts. Lipids belonging to PCs, PIs, SMs, DAG, TAG, PS, PA, and PG classes were identified and their distribution over the xenograft was determined. Three areas were identified in the xenograft, corresponding to cells in different metabolic stages and to a layer of adipose tissue that covers the xenograft.

On line characterization of 58 phenolic compounds in Citrus fruit juices from Spanish cultivars by high-performance liquid chromatography with photodiode-array detection coupled to electrospray ionization triple quadrupole mass spectrometry.

Polyphenol profile of Citrus juices of sweet orange, tangerine, lemon and grapefruit from Spanish cultivars was obtained by High-Performance Liquid Chromatography with Diode Array Detection coupled to Electrospray ionization and Triple Quadrupole Mass Spectrometry. Fifty eight phenolic compounds of five different classes were identified in these Citrus juices. Flavanone: O-dihexoside of naringenin; flavones: apigenin-7-O-rutinoside-4'-O-glucoside, luteolin-7-O-neohesperidoside-4'-O-glucoside, luteolin-6-C-glucoside, 6,8-di-C-acylhexosides of chrysoeriol and diosmetin, 6C- and 8C-glucoside-O-pentoside of apigenin, apigenin-6-C-hexoside-O-hexoside and apigenin-8-C-hexoside-O-acylrhamnoside; flavonols: 7-O-rutinosides of quercetin, kaempferol, isorhamnetin and tamarixetin, kaempferol-3-O-rutinoside, isorhamnetin-3-O-rutinoside-7-O-glucoside, tamarixetin-3-O-rutinoside-7-O-glucoside, isorhamnetin-3-O-hexoside-7-O-rhamnosylhexoside, 3-O-rhamnoside-7-O-rhamnosylhexoside of quercetin and isorhamnetin and kaempferol-3-O-rhamnosylhexoside-7-O-rhamnoside; hydroxycinnamic acids: O-hexoside of ferulic and sinapic acid; and, coumarins: O-hexoside and O-rhamnosylhexoside of scopoletin, had not previously been reported in Citrus juices to our knowledge. Structures have been assigned on the basis of the complementary information obtained from retention time, UV-visible spectra, scan mode MS spectra, and fragmentation patterns in MS(2) spectra obtained using different collision energies. A structure diagnosis scheme is provided for the identification of different phenolic compounds.

Chemometric characterization of fruit juices from Spanish cultivars according to their phenolic compound contents: I. Citrus fruits.

The data set composed by phenolic compound profiles of 83 Citrus juices (determined by HPLC-DAD-MS/MS) was evaluated by chemometrics to differentiate them according to Citrus species (sweet orange, tangerine, lemon, and grapefruit). Cluster analysis (CA) and principal component analysis (PCA) showed natural sample grouping among Citrus species and even the Citrus subclass. Most of the information contained in the full data set can be captured if only 15 phenolic compounds (concentration ≥10 mg/L), which can be quantified with fast and accurate methods in real samples, are introduced in the models; a good classification which allows the confirmation of the authenticity of juices is achieved by linear discriminant analysis. Using this reduced data set, fast and routine methods have been developed for predicting the percentage of grapefruit in adulterated sweet orange juices using principal component regression (PCR) and partial least-squares regression (PLS). The PLS model has provided suitable estimation errors.

A general analytical strategy for the characterization of phenolic compounds in fruit juices by high-performance liquid chromatography with diode array detection coupled to electrospray ionization and triple quadrupole mass spectrometry.

In the present study, a methodology based on liquid chromatography with diode array detection (HPLC/DAD) coupled to an electrospray ionization (ESI) interface and a triple quadrupole mass spectrometer for the simultaneous identification of phenolic compounds in fruit juices has been developed. 72 available phenolic compound standards from diverse families present in fruits have been studied in order to analyze their fragmentation pattern. As a result, a general strategy for the characterization of unknown phenolic compounds in fruit juices was designed: (i) taking into account its UV-visible spectrum and elution order, assign the unknown polyphenol to a polyphenol class, (ii) identify the quasi-molecular ion using positive and negative MS spectra, being supported by adducts generated with solvent or sodium and molecular complexes, (iii) determinate the pattern of glycosylation in positive mode using ESI(+)-CID MS/MS product ion scan experiments, selecting the quasi-molecular ion as precursor ion, and finally, (iv) study the identity of the aglycone through ESI(+)-CID MS/MS product ion spectra from the protonated aglycone, [Y(0)](+). This strategy was successfully employed for the characterization of known and unknown phenolic compounds in juices from 17 different fruits.

New features on the fragmentation and differentiation of C-glycosidic flavone isomers by positive electrospray ionization and triple quadrupole mass spectrometry.

Six flavone mono-C-glucosides, four standards (beta-D-glucopyranosyl-(1 --> C-6)- and -(1 --> C-8)- apigenin and luteolin) and two others from lemon juice (beta-D-glucopyranosyl-(1 --> C-6)- and -(1 --> C-8)-diosmetin) have been studied in order to analyze their fragmentation patterns. Initial separation was carried out using high-performance liquid chromatography with diode-array detection (HPLC/DAD) coupled to an electrospray ionization (ESI) interface and a triple quadrupole mass spectrometer. Several systematic differences between collision-induced dissociation tandem mass (CID-MS/MS) spectra of C-6- and C-8-isomers have been found and some general guidelines and two new diagnostic product ions have been proposed for the differentiation of C-6- and C-8-flavonoid glycosides. These results have been successfully applied to the characterization of two flavone C-glycosides found in lemon juice, and mass spectra of a flavone di-C-glycoside detected in lemon juice have been studied and interpreted.