Nanoliposomes were first used to encapsulate two fluorophores, fluorescein and cresyl violet, to construct a FRET-based ratiometric fluorescent probe, for sensing intracellular pH values. The morphology, stability, interference factors, biocompatibility, and performance were investigated in detail. The fluorophores-encapsulated nanoliposomes (FEN) probe can be uptaken by cells within 30 min, which is faster than any other nanosensor, and cell viability was not significantly changed upon treatment with the FEN probe for 24 h, showing the low cytotoxicity and good biocompatibility of the probe. Quantitative determinations of intracellular pH of MCF-7 cells and the pH fluctuations associated with inflammation have been successfully performed with our probe. This work demonstrated that nanoliposomes could be served as an emerging approach for constructing useful fluorescent nanosensors with low toxicity.

•Triacylglycerol (TG) with specific fatty acid chain were qualified and quantified.•Multiple MS modes of QTRAP MS and FT-ICR MS were combined for the TG analysis.•The isomer TGs interferences and the isobaric interference were indicated and avoided.•Many oxidized TG species were identified.•TGs with the degree of unsaturation >3 were significantly decreased in HCC tissue.High levels of triacylglycerols (TGs) have been linked to cardiovascular disease and liver diseases. Comprehensively analyzing TGs is helpful to understand the TGs functions in these diseases. However, due to the existence of a large number of isomers TGs and the lack of commercial standards, precise analysis of individual triacylglycerol (TG) with specific fatty acid chain composition is full of challenge. In this work, ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization (ESI) mass spectrometry (MS) were employed for comprehensive qualification and quantification of TGs with specific fatty acid chain composition in horse adipose tissue, human plasma and liver tissues including hepatocellular carcinoma (HCC) and para-carcinoma tissues. Multiple MS detection modes from QTRAP MS and FT-ICR MS were utilized, and hundreds of TG species (including many oxidized TG species) with their specific fatty acid chain compositions have been qualified and quantified. The isomer TGs interference, the isobaric interference, and oxidized TG species interference were firstly indicated. Several isomer TGs, for example, 18:1/20:1/18:2 TG and 20:3/18:1/18:0 TG, which were all 56:4 TG, demonstrated different trends in HCC tissue compared with para-carcinoma tissue, which showed the importance of analysis of TG with specific fatty acid chain composition. In addition, 10 TGs with the degree of unsaturation beyond three were significantly decreased, while 16:0/17:0/18:0 TG, no double bond, was significantly increased in the HCC tissue, which firstly revealed aberrant specific TG metabolism in HCC. This is a systematic report about comprehensive analysis of TGs by UPLC-ESI-MS, which is of significance for accurate analysis of these lipids.Download high-res image (114KB)Download full-size image

•A nontargeted lipidomics approach for potential diagnostic biomarkers in plasma of OC patients.•LPCs were up-regulated, and PCs and TGs were down-regulated in plasma of OC patients.•Glycerophospholipid metabolism as a key pathway that was disregulated in the disease.Ovarian cancer (OC) is the most common cause of death from gynecologic malignancies in women. The identification of reliable diagnostic biomarkers for the early detection of this deadly disease is critical for reducing the mortality rate of OC. Plasma lysophosphatidic acid (LPA) levels were increased from OC patients vs. healthy controls. Therefore, lipidomics may represent an excellent developing prospect for the discovery of diagnostic biomarkers of OC. In this study, a nontargeted lipidomics approach based on ultra performance liquid chromatography-electrospray ionization-QTOF-mass spectrometry (UPLC-ESI-QTOF-MS) combined with multivariate data analysis, including principal component analysis (PCA) and (orthogonal) partial least squared discriminant analysis [(O)PLS-DA] was applied for the investigation of potential diagnostic biomarkers in plasma of OC patients. Patients with OC could be distinguished from healthy individuals and patients with benign gynecological tumor disease by this method, which shows a significant lipid perturbation in this disease. With the assistance of high resolution and high accuracy of MS and MS/MS data, the potential markers including lysophosphatidylcholines (LPCs), phosphatidylcholines (PCs) and triacylglycerols (TGs) with specific fatty acid chains, were identified. Interestingly, LPCs were up-regulated and PCs and TGs were down-regulated, compared OC group with benign tumor and normal control groups, and the glycerophospholipid metabolism emerged as a key pathway, in particular, the phospholipase A2 (PLA2) enzyme activity, that was disregulated in the disease. This study may provide new insight into underlying mechanisms for OC and proves that MS-based lipidomics is a powerful method in discovering new potential clinical biomarkers for diseases.

Despite huge advances in the research of epithelial ovarian cancer (EOC), it remains the most lethal gynecological malignancy. Peritoneal tumor cell dissemination with cell survival and drug-resistance to taxane and platinum-based chemotherapy are two of the major challenges of EOC treatment. We have generated highly aggressive EOC cell lines (ID8-P1 lines or P1) from ID8-P0 (without in vivo passage, or P0) through in vivo passage in mice. We conducted lipidomic analyses in cells from ID8-P0 versus three ID8-P1 cell lines using ultra-high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. A total of 16 classes of lipids (149 individual lipids) were analyzed and compared between P0 and P1 cells. In addition to overall lipid profiles in EOC cells, we had several novel observations. Several classes and species of lipids have been identified to be differentially present in P0 versus P1 cells, which are potentially involved in the acquired aggressiveness of P1 cells. Triacylglycerols (TAG) were dramatically increased under detachment stress in EOC cells. Since survival of EOC cells under detachment is one of the major obstacles for EOC treatment, further studies identifying the molecular mechanisms controlling TAG increase may lead to new treatment modalities for EOC.

•Optimization of temperature improved the response of heat labile compound, e.g. ST.•The isobaric interferences were widespread phenomenon in MS analysis of lipids.•A baseline separation of conflicting species by UHPLC was necessary.•Many lipids were significantly changed in drug resistance tumor cells.•PLA2 and GCS enzymes and their regulated lipids were related to drug resistance.Glycerophospholipids and sphingolipids are important signaling molecules which are involved in many physiological and pathological processes. Here we reported an effective method for accurate analysis of these lipids by liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS). The methanol method was adopted for extraction of lipids due to its simplicity and high efficiency. It was found that two subclasses of sphingolipids, sulfatide (ST) and cerebroside (CB), were heat labile, so a decreased temperature in the ion source of MS might be necessary for these compounds analysis. In addition, it was found that the isobaric interferences were commonly existent, for example, the m/z of 16:0/18:1 PC containing two 13C isotope being identical to that of 16:0/18:0 PC determined by a unit mass resolution mass spectrometer; therefore, a baseline separation of interferential species was required to maintain selectivity and accuracy of analysis. In this work, an ultra-high-performance liquid chromatography (UHPLC)-based method was developed for separation of interferential species. Moreover, in order to deal with the characteristics of different polarity and wide dynamic range of glycerophospholipids and sphingolipids in biological systems, three detecting conditions were combined together for comprehensive and rational analysis of glycerophospholipids and sphingolipids. The method was utilized to profile glycerophospholipids and sphingolipids in drug resistant tumor cells. Our results showed that many lipids were significantly changed in drug resistant tumor cells compared to paired drug sensitive tumor cells. This is a systematic report about the isobaric interferences and heat labile compounds interferences when analyzing glycerophospholipids and sphingolipids by ESI-MS/MS, which aids in ruling out one potential source of systematic error to ensure the accuracy of analysis.

A very uniform 2,5-dihydroxybenzoic acid (DHB) layer was for the first time constructed and used as a matrix for matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS) for quickly exploring the changes in lipids within biological systems. Lipid extracts from biological samples were dissolved in chloroform and deposited onto the DHB layer. Benefiting from the insolubility of DHB in chloroform, the uniform matrix crystals were still maintained, and more importantly, the lipid analytes were distributed homogeneously on the layer, which significantly increased the reproducibility of analysis using MALDI-FTICR MS. Taking advantage of the benefit of high resolution of FTICR MS and the fragment ions obtained by MS/MS, lots of lipids were identified. This method was used for exploring the changes of lipids in drug-resistant tumor cells compared with paired drug-sensitive tumor cells. The principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were employed for discovery of the changed lipids. This method, characterized by the simplicity and the speediness, demonstrated a new and promising approach for lipidomics study.

The quantitative analysis by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) is a challenge due to the poor reproducibility originating from the heterogeneity of the matrix–analyte crystals. Polystyrene (PS) colloidal spheres have superior monodispersed property and can self-assemble to form photonic crystals. With the assistance of PS spheres, a uniform matrix–analyte cocrystal was constructed for the quantitative analysis of plasma lysophosphatidylcholines (LPCs). The matrix and the solvent in MALDI MS analysis were optimized, and the reproducibility and the accuracy were investigated in detail. This is the first report about the very simple method of PS spheres-assisted MALDI MS for quantitative analysis, where it is believed that this approach will greatly expand the applications of MALDI MS.