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Infections are still among the most important causes of morbidity and mortality in patients with lung cancer, which has the highest rate of cancer-related deaths in the world. Microsporidia, which are opportunistic parasitic fungi, primarily localize to the intestine by ingestion but can disseminate to the respiratory tract or can be acquired by spore inhalation. Cancer patients are at higher risk for microsporidia, a life-threatening infection, than the normal population is. We aimed to characterize the prevalence of microsporidia infection for the first time by evaluating the intestinal and respiratory tracts of patients with lung cancer. In this study, we investigated 98 patients with lung cancer and 103 healthy individuals for microsporidia infection and evaluated the clinical findings of patients who were found to be positive. Sputum and stool samples were tested by microscopic examination, in addition to pan-microsporidia and genus-specific polymerase chain reactions. Nine patients with lung cancer had positive results for microsporidia (9.2%), which was significantly higher than the rate in healthy individuals (P = 0.008), and most of them had clinical findings. Among these positive patients, polymerase chain reaction revealed microsporidia in the sputum samples of seven patients, the stool sample of one patient, and both the sputum and stool samples of one patient. Encephalitozoon cuniculi was identified as the predominant pathogen in 87.5% (7/8) of positive sputum samples. Microsporidia infection was significantly associated with advanced stages of cancer. However, in the control group, Encephalitozoon intestinalis was detected in the stool sample of an individual without clinical symptoms. Microsporidia, especially E. cuniculi, should be considered as a cause of respiratory tract infection as well as intestinal infection in cancer patients and should be screened in respiratory samples of these patients when they have pulmonary symptoms.

Extracellular vesicles (EVs) are cell-derived membrane-bound particles, including exosomes and microvesicles that differ in cellular origin, content, and lipid composition. This study reports that exosomes and microvesicles can be simultaneously separated by size using flow field-flow fractionation (FlFFF) employed with field programming and that the detection of low-concentration EV species can be significantly improved using multiangle light scattering (MALS). The efficiency of ultracentrifugation (UC) and ultrafiltration (UF) in isolating EVs from the culture media of DU145 cells was compared, and the results showed that UF retrieves more EVs than UC. Two size fractions (small and large) of both exosomes and microvesicles were collected during the FlFFF runs and examined using Western blotting to confirm each EV, and transmission electron microscopy was performed for size analysis. Sizes were compared using the root-mean-square radius obtained from the MALS calculation. The collected fractions were further examined using nanoflow ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry for the size-dependent lipidomic profiles of exosomes and microvesicles, showing that lipids were more enriched in the fraction containing large exosomes than in that containing small exosomes; however, an opposite trend was observed with microvesicles. The present study demonstrated that UF followed by FlFFF-MALS can be utilized for the size separation of exosomes and microvesicles without sequential centrifugation, which is useful for monitoring the changes in the size distribution of EVs depending on the biological status along with generating size-dependent lipidomic profiles.

Supplementary Table 1, Supplementary Figure 1 from Systematic Error in Gas Chromatography-Mass Spectrometry–Based Quantification of Hydrolyzed Urinary Steroids

Gas chromatography-mass spectrometry–based metabolite profiling can lead to an understanding of various disease mechanisms as well as to identifying new diagnostic biomarkers by comparing the metabolites related in quantification. However, the unexpected transformation of urinary steroids during enzymatic hydrolysis with Helix pomatia could result in an underestimation or overestimation of their concentrations. A comparison of β-glucurondase extracted from Escherichia coli revealed 18 conversions of 84 steroids tested as an unexpected transformation under hydrolysis with β-glucuronidase/arylsulfatase extracted from Helix pomatia. In addition to the conversion of 3β-hydroxy-5-ene steroids into 3-oxo-4-ene steroids, which has been reported, the transformation of 3β-hydroxy-5α–reduced and 3β-hydroxy-5β–reduced steroids to 3-oxo-5α–reduced and 3-oxo-5β–reduced steroids, respectively, was newly observed. The formation of by-products was in proportion to the concentration of substrates becoming saturated against the enzyme. The substances belonging to these three steroid groups were undetectable at low concentrations, whereas the corresponding by-products were overestimated. These results indicate that the systematic error in the quantification of urinary steroids hydrolyzed with Helix pomatia can lead to a misreading of the clinical implications. All these hydrolysis procedures are suitable for study purposes, and the information can help prevent false evaluations of urinary steroids in clinical studies. Cancer Epidemiol Biomarkers Prev; 19(2); 388–97

This study introduces the thickness-tapered channel design for flow field-flow fractionation (FlFFF) for the first time. In this design, the channel thickness linearly decreases along the channel axis such that the flow velocity increases down the channel. Channel thickness is an important variable for controlling retention time and resolution in field-flow fractionation. Especially, in the steric/hyperlayer mode of FlFFF, in which particles (1 μm) migrate at elevated heights above the channel wall owing to hydrodynamic lift forces, the migration of long-retaining smaller-sized particles can be enhanced in a relatively thin channel or by increasing the migration flow rate; however, an upper size limit that can be resolved is simultaneously sacrificed. A thickness-tapered channel was constructed without a channel spacer by carving the surface of a channel block such that the channel inlet was deeper than the outlet (

Lipids are crucial components of cellular function owing to their role in membrane formation, intercellular signaling, energy storage, and homeostasis maintenance. In the brain, lipid dysregulations have been associated with the etiology and progression of neurodegeneration and other neurological pathologies. Hence, brain lipids are emerging as important potential targets for the early diagnosis and prognosis of neurological diseases. This review aims to highlight the significance and usefulness of lipidomics in diagnosing and treating brain diseases. We explored lipid alterations associated with brain diseases, paying attention to organ-specific characteristics and the functions of brain lipids. As the recent advances in brain lipidomics would have been impossible without advances in analytical techniques, we provide up-to-date information on mass spectrometric approaches and integrative analysis with other omic approaches. Last, we present the potential applications of lipidomics combined with artificial intelligence techniques and interdisciplinary collaborative research for treating brain diseases with clinical heterogeneities.

Graphene oxide (GO)-a chemical derivative of graphene with numerous oxygen functional groups on its surface-has attracted considerable interest because of its intriguing properties in relation to those of pristine graphene. In addition to the inherent wide lateral size distribution of GO sheets arising from the typical oxidative exfoliation of graphite, control of the lateral size of GO is critical for desired GO-based applications. Herein, flow/hyperlayer field-flow fractionation (flow/hyperlayer FFF) is optimized to separate GO sheets by lateral dimensions. Optimized fractionation is achieved by investigating the influences of carrier solvent, channel thickness, and flow rate conditions on the steric/hyperlayer separation of GO sheets by flow FFF. Due to the strong hydrodynamic lift forces of extremely thin GO sheets, a thick flow FFF channel (w = 350 μm) and a very low field strength are required to retain the GO sheets within the channel. GO sheets with narrow size fractions are successfully collected from two different graphite sources during flow/hyperlayer FFF runs and are examined to verify the size evolution. Considering the average lateral diameter of the GO fraction calculated on the basis of the assumption of a circular disk shape, the retention of the GO sheets is 2.2-5.0 times faster than that of spherical particles of the same diameter. This study demonstrates that through flow/hyperlayer FFF, the size distribution of GO sheets can be determined and narrow size fractions can be collected (which is desirable for GO-based applications), which are commonly influenced by the GO lateral dimension.

Due to the increased applications of engineered elemental metal nanoparticles (EMNPs) in recent years, increased attention has been devoted to their release into the environment. EMNPs pose potenti...

The gut microbiome can influence the development of tumours and the efficacy of cancer therapeutics1-5; however, the multi-omics characteristics of antitumour bacterial strains have not been fully elucidated. In this study, we integrated metagenomics, genomics and transcriptomics of bacteria, and analyses of mouse intestinal transcriptome and serum metabolome data to reveal an additional mechanism by which bacteria determine the efficacy of cancer therapeutics. In gut microbiome analyses of 96 samples from patients with non-small-cell lung cancer, Bifidobacterium bifidum was abundant in patients responsive to therapy. However, when we treated syngeneic mouse tumours with commercial strains of B. bifidum to establish relevance for potential therapeutic uses, only specific B. bifidum strains reduced tumour burden synergistically with PD-1 blockade or oxaliplatin treatment by eliciting an antitumour host immune response. In mice, these strains induced tuning of the immunological background by potentiating the production of interferon-γ, probably through the enhanced biosynthesis of immune-stimulating molecules and metabolites.

Many interesting properties of 2D materials and their assembled structures are strongly dependent on the lateral size and size distribution of 2D materials. Accordingly, effective size separation of polydisperse 2D sheets is critical for desirable applications. Here, we introduce flow field-flow fractionation (FlFFF) for a wide-range size fractionation of graphene oxide (GO) up to 100 μm. Two different separation mechanisms are identified for FlFFF, including normal mode and steric/hyperlayer mode, to size fractionate wide size-distributed GOs while employing a crossflow field for either diffusion or size-controlled migration of GO. Obviously, the 2D GO sheet reveals size separation behavior distinctive from typical spherical particles arising from its innate planar geometry. We also investigate 2D sheet size-dependent mechanical and electrical properties of three different graphene fibers produced from size-fractionated GOs. This FlFFF-based size selection methodology can be used as a generic approach for effective wide-range size separation for 2D materials, including rGO, TMDs, and MXene.

Exosomes are extracellular vesicles that mediate intercellular communication, immune response, and tumour metastasis. However, exosome isolation from the blood is complicated because their size and density are similar to those of blood lipoproteins. Here, we employed field programming frit-inlet asymmetrical flow field-flow fractionation (FIAF4) coupled with multiangle light scattering (MALS) for the effective separation of exosomes from free unbound proteins and lipoproteins present in serum samples using different pre-treatment methods, namely, a commercial exosome isolation kit, ultracentrifugation (UC), and a simple centrifugation followed by ultrafiltration (UF). Sizes of the eluted exosomes, as calculated by MALS signals, approximated well with the results of batch dynamic light scattering of the collected fractions and with the sizes of polystyrene particles. Exosome separation from lipoproteins was validated by western blotting with several markers of exosomes and lipoproteins, followed by proteomic analysis using nanoflow ultrahigh-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry. UC requires relatively large amounts of serum samples (at least 2 mL) but is more efficient at removing lipoproteins. The UF method with a centrifugal concentrator (300 kDa) was found to be more effective in retrieving exosomes with low serum volumes (50 μL). Altogether, this study demonstrates the application of field programming FIAF4 for the isolation/purification of exosomes from proteins and lipoproteins in the serum.

Saliva is a readily accessible and clinically useful biofluid that can be used to develop disease biomarkers because of a variety of biologically active molecules in it that are also found in blood. However, even though saliva sampling is simple and non-invasive, few studies have investigated the use of salivary lipids as biomarkers, and the extraction of lipids from saliva needs to be examined thoroughly. In the present study, methods (i.e., saliva sample volume, 0.1-1.0 mL) for the extraction and analysis of salivary lipids by nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry (nUHPL-CESI-MS/MS) were evaluated according to the matrix effect, extraction recovery, and number of quantifiable lipids. A total of 780 lipids were identified in a pooled saliva sample from 20 healthy volunteers, and 372 lipids without differentiating acyl chain structures were quantified, along with comprehensive information on salivary lipid composition and individual lipid levels. Even though extraction recovery was maintained at saliva sample volumes as low as 0.2 mL, the matrix effect and limit of detection (LOD) were relatively large with 1.0 mL. Considering the matrix effect, LOD, and number of quantifiable lipids (>limit of quantitation), the minimum volume of saliva sufficient for lipidomic analysis using nUHPLC-ESI-MS/MS was determined to be 0.5 mL. (C) 2021 Elsevier B.V. All rights reserved., National Research Foundation (NRF) of Korea [NRF-2020K2A9A1A06097918]; [NRF-2021R1A2C2003171], Role of the funding source: This work was supported by a bilateral research program (NRF-2020K2A9A1A06097918) between Turkey and Korea supported by the National Research Foundation (NRF) of Korea and in part by NRF-2021R1A2C2003171.

We investigated the effect of oxidative stress (OS) on lipidomic perturbations in the subcellular fractions and exosomes of human embryonic kidney (HEK) 293 cells using asymmetrical flow field–flow fractionation (AF4) and nanoflow ultrahigh performance liquid chromatography–electrospray ionization–tandem mass spectrometry (nUHPLC-ESI-MS/MS). We treated HEK 293 cells with hydrogen peroxide (H2O2) and fractionated the cell lysates using AF4 to determine the change in size and population of the subcellular fractions and exosomes, and to obtain narrow size fractions for lipid analysis. A total of 438 lipids from 642 identified species—including oxidized lipids—were quantified. The relative amount of secreted exosomes increased by 28% during OS, whereas the amount of subcellular species decreased by 35%. There was a significant increase in the level of oxidized phospholipids in the mitochondrion-enriched subcellular fractions, but not in the exosomes. Most high-abundance triacylglycerol (TG) species increased in the stressed cells, whereas they decreased in the exosomes. During OS, ceramides involved in the apoptotic mitochondrial pathway were accumulated in the subcellular fractions, whereas their levels were unaffected in the exosomes. The present study demonstrated that AF4 and nUHPLC-ESI-MS/MS can be used to investigate lipid alterations in subcellular and extracellular species during OS, and the pathological relationships in diseases caused by reactive oxygen species.

Flow field-flow fractionation (FlFFF) is a versatile size-based separation method suitable for biological macromolecules including proteins/protein aggregates, DNA, subcellular organelles, extracellular species, and whole cells. This review introduces briefly the basic principles of FlFFF and its recent applications for proteomic and lipidomic analysis, which are described in two parts: (1) off-line coupling of FlFFF with MS and other bioanalytical methods, and (2) on-line FlFFF with MS. The first part includes applications for lipoproteins, exosomes, and subcellular organelles for the size-dependent analysis of proteins and lipids in narrow size-fractions collected during FlFFF, followed by independent analysis including western blotting and nanoflow liquid chromatography-electrospray ionisation-tandem mass spectrometry (nLC-ESI-MS/MS). The second part highlights the on-line FlFFF-MS, in which a miniaturised FlFFF channel is coupled to ESI-MS/MS for the high-speed lipid analysis of lipoproteins and to inductively-coupled plasma MS for the direct analysis of metals in metalloproteins from blood plasma.

A comprehensive lipidomic analysis at the molecular level using nanoflow ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (nUHPLC-ESI-MS/MS) was performed to elucidate the lipid profiles of patient blood samples from five commonly found cancers (liver, lung, gastric, colorectal, and thyroid), which were then compared with the lipid profiles of healthy controls. From a total of 335 lipids identified and quantified, 50 high abundance lipids showing significant changes (2-fold and p 0.01) in at least one of the five cancers (vs. controls) were analysed. Lipid species were found to be significantly associated with more than one type of cancer; the numbers of lipid species found as significantly changed in all five, four, three, two, and one type of cancer were 1, 8, 8, 15, and 17, respectively. Among these, the high abundance phosphatidylethanolamine species, including lysophosphatidylethanolamine and PE plasmalogen, was significantly low in four cancer types, but was high in thyroid cancer. Receiver operating characteristic analysis resulted in the selection of lipids specific to each cancer: liver (four phosphatidylinositols and diacylglycerol 16:1_18:0), gastric (phosphatidylcholine 34:2, 36:3, and 36:4, and lysophosphatidic acid 18:2), lung (lysophosphatidylinositol 16:0, sphingomyelin d18:1/20:0, and triacylglyceride 50:1 and 54:4), and thyroid (lysophosphatidylinositol 18:0 and 18:1). Our results provide a basis for future validation of cancer-specific lipid markers with high diagnostic ability.

Herein, we introduce a comprehensive analytical method for the separation and relative quantification of polyglycerophospholipids (PGPLs), including phosphatidylglycerol (PG), bis(monoacylglycero)phosphate (BMP), bis(diacylglycero)phosphate (BDP), Hemi BDP, cardiolipin (CL), monolysocardiolipin (MLCL), and dilysocardiolipin (DLCL), using isotope-labeled methylation (ILM) with nanoflow ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (nUHPLC-ESI-MS/MS). Abnormal levels of BMP and CL have been associated with the pathology of lysosomal storage and neurodegenerative diseases. Thus, simultaneous analysis of all PGPLs is important to understand the mechanisms and pathologies of such diseases. In this study, improved separation and MS detection of PGPLs, including their regioisomers, was achieved by the methylation of PGPL. ILM-based relative quantification was applied to lipid extracts from a dopaminergic cell line (SH-SY5Y) treated with drugs commonly used for Parkinson's disease (PD), resulting in the identification of 229 unique PGPLs, including 121 CLs, 71 MLCLs, and 16 Hemi BDP species. The drug treatment induced significant increases in the amount of CLs containing polyunsaturated fatty acyl chains, including 20:4 and 22:6, as well as decreased levels of BMP, Hemi BDP, and BDP species, demonstrating the feasibility of using ILM for the comprehensive and high-speed relative quantification of PGPLs.

This study introduces a high-speed screening method for the quantitative analysis of lipoprotein components in human plasma samples using online miniaturized asymmetrical flow field-flow fractionation and electrospray ionization-tandem mass spectrometry (mAF4-ESI-MS/MS). Using an mAF4 channel, high-density lipoproteins and low-density lipoproteins can be fractionated by size at a high speed (

Lipid analysis is a powerful tool that can elucidate the pathogenic roles of lipids in metabolic diseases, and facilitate the development of potential biomarkers. Lipid analysis by large-scale lipidomics requires a high-speed and high-throughput analytical platform. In the present study, a high-speed analytical method for lipid analysis using nanoflow ultrahigh-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry (nUHPLC-ESI-MS/MS) was optimised by investigating the effects of column flow rate, pump flow rate, dwell time, initial binary mobile phase composition, and gradient duration on the separation efficiency of standard lipid mixtures. The minimum gradient time for high-speed lipid separation was determined by examining the time-based separation efficiency and spectral overlap of isobaric lipid species during selected reaction monitoring-based quantification of sphingomyelin and a second isotope of phosphatidylcholine, which differ in molecular weight by only 1 Da. Finally, the optimised nUHPLC-ESI-MS/MS method was applied to analyse 200 plasma samples from patients with liver, gastric, lung, and colorectal cancer to evaluate its performance by measuring previously identified candidate lipid biomarkers. About 73% of the reported marker candidates (6 out of 7 in liver, 5/9 in gastric, 4/6 in lung, and 6/7 in colorectal cancer) could be assigned using the optimised method, supporting its use for high-throughput lipid analysis.

Background Clinical diagnosis of post-hepatectomy liver failure (PHLF) can only be made on or after the 5th postoperative day. Biomarker for early diagnosis is considered as a critical unmet need. Methods Twenty domestic female crossbreed (Yorkshire-landrace and duroc) pigs underwent sham operation (n=6), 70% (n=7) and 90% (n=7) partial hepatectomy (PH). A comprehensive lipidomic analysis was conducted using sera collected at pre-operation (PO), 14, 30, and 48 h after PH using nanoflow ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry. Results Of the 184 quantified lipids, 14 lipids showed significant differences between the two resection groups starting at 30 h after surgery. Four phosphatidylcholine (PC) plasmalogen species (P-16:0/16:0, P-18:0/18:2, P-18:0/20:4, and P-18:0/22:6) and PC 32:2 significantly increased in the 90% PH group while these returned to PO level after 30 h in the 70% PH group, presumably implying the failure markers. In contrast, eight triacylglycerol (TG) species (40:0, 42:1, 42:0, 44:1, 44:2, 46:1, 46:2, and 48:3) and sphingomyelin d18:1/20:0 showed an opposite trend, wherein they significantly decreased in the 90% PH group while these in the 70% PH group were abruptly increased until 30 h but returned to near PO levels at 48 h, implying the recovery markers. Same trends could also be observed in the level of whole lipid classes of PC plasmalogens and TGs, in addition to selected individual lipid species. Conclusions Characteristic lipidomic signatures of PHLF could be identified using large animal models. These candidates have a potential to serve as a tool for early diagnosis and may open new paths to the study to overcome PHLF.

Daily physical exercise is an essential part of life and is required for remaining healthy; it enhances therapeutic efficacy in the elderly and prevents age-related diseases associated with lipid profile alterations, such as cardiovascular disease, diabetes mellitus, and dementia. To more efficiently analyse the lipid profiles and unveil the effect of exercise in aged mice, we optimized our study by examining the effects of using ionization modifiers in the mobile phase and in-source fragmentation of lysophospholipids on the simultaneous analysis of fatty acids (FAs) including hydroxyl fatty acids, glycerophospholipids, sphingolipids, and glycerolipids using nanoflow ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry. We applied the optimization to investigate the lipidomic plasma alterations in young (7 weeks old) and aged (84 weeks old) mice (C57BL/6) subjected to treadmill exercise. Of the 390 identified lipid species, 159 were quantified to investigate ageing-related lipid species responsive to physical exercise. In particular, circulating lysophosphatidylcholine and lysophosphatidylethanolamine levels showed a significant decrease, and lysophosphatidic acid showed a simultaneous increase with ageing. The saturated FA (16:0 and 18:0) increased with ageing while the unsaturated FA 22:6 decreased. Dihydroxy fatty acid (18:1_2OH) showed an exercise-induced recovery against ageing. It is notable that the levels of five triacylglycerol species significantly increased by as much as threefold with ageing, but their levels largely recovered to those observed in the young mice after exercise. These findings can help understand the influence of ageing on lipid perturbation and the role of physical exercise on lipidomic recovery in response to ageing-associated loss of physical status. Graphical abstract.

Transition metals in airborne particulate matter, especially those with aerodynamic diameters no more than 2.5 μm (PM

Osteoporosis, a degenerative bone disease characterized by reduced bone mass and high risk of fragility, is associated with the alteration of circulating lipids, especially oxidized phospholipids (Ox-PLs). This study evaluated the lipidomic changes in lipoproteins of patients with postmenopausal osteoporosis (PMOp) vs. postmenopausal healthy controls. High-density lipoproteins (HDL) and low-density lipoproteins (LDL) from plasma samples were size-sorted by asymmetrical flow field-flow fractionation (AF4). Lipids from each lipoprotein were analyzed by nanoflow ultrahigh performance liquid chromatography&ndash, electrospray ionization&ndash, tandem mass spectrometry (nUHPLC&ndash, ESI&ndash, MS/MS). A significant difference was observed in a subset of lipids, most of which were increased in patients with PMOp, when compared to control. Phosphatidylethanolamine plasmalogen, which plays an antioxidative role, was increased in both lipoproteins (P-16:0/20:4, P-18:0/20:4, and P-18:1/20:4) lysophosphatidic acid 16:0, and six phosphatidylcholines were largely increased in HDL, but triacylglycerols (50:4 and 54:6) and overall ceramide levels were significantly increased only in LDL of patients with PMOp. Further investigation of 33 Ox-PLs showed significant lipid oxidation in PLs with highly unsaturated acyl chains, which were decreased in LDL of patients with PMOp. The present study demonstrated that AF4 with nUHPLC&ndash, MS/MS can be utilized to systematically profile Ox-PLs in the LDL of patients with PMOp.

The gut microbiome can influence the development of tumours and the efficacy of cancer therapeutics

Retention in flow field-flow fractionation (flow FFF) is generally governed by the combination of crossflow and migration flowrates. Especially for an asymmetrical flow FFF (AF4) channel in which the channel-inlet flow is divided into crossflow and outflow, the separation of low-molecular-weight proteins or macromolecules requires a relatively high crossflow rate along with a very low outflow rate for a reasonable level of resolution, which often leads to a limitation in channel pressure. In this study, the performances of AF4 with increased channel thicknesses have been investigated by adjusting the effective channel flowrates in the asymmetrical channels according to the variation of channel thickness. Four AF4 channels of different channel thicknesses (350, 490, 600, and 740 μm) were employed to examine the potential usefulness of employing a thick channel in the high-resolution separation of low-molecular-weight proteins (100 kDa) and to determine the relationship between higher channel thickness and the recovery of elution. Experiments showed that the ratio of crossflow rate to the effective channel flowrate should be considered in the selection of a run condition at an increased channel thickness. The study also demonstrated that a thick AF4 channel can be useful for the high-resolution separation of low-molecular-weight species such as protein aggregates without using extremely high crossflow rates.

Given their important role in neuronal function, there has been an increasing focus on altered lipid levels in brain disorders. The effect of a high-fat (HF) diet on the lipid profiles of the cortex, hippocampus, hypothalamus, and olfactory bulb of the mouse brain was investigated using nanoflow ultrahigh pressure liquid chromatography-electrospray ionization-tandem mass spectrometry in the current study. For 8 weeks, two groups of 5-week-old mice were fed either an HF or normal diet (6 mice from each group analyzed as the F and N groups, respectively). The remaining mice in both groups then received a 4-week normal diet. Each group was then subdivided into two groups for another 4-week HF or normal diet. Quantitative analysis of 270 of the 359 lipids identified from brain tissue revealed that an HF diet significantly affected the brain lipidome in all brain regions that were analyzed. The HF diet significantly increased diacylglycerols, which play a role in insulin resistance in all regions that were analyzed. Although the HF diet increased most lipid species, the majority of phosphatidylserine species were decreased, while lysophosphatidylserine species, with the same acyl chain, were substantially increased. This result can be attributed to increased oxidative stress due to the HF diet. Further, weight-cycling (yo-yo effect) was found more critical for the perturbation of brain lipid profiles than weight gain without a preliminary experience of an HF diet. The present study reveals systematic alterations in brain lipid levels upon HF diet analyzed either by lipid class and molecular levels.

Alzheimer's disease (AD) is an irreversible neurodegenerative disorder with the clinical symptom of the progressive loss of cognitive function and mild cognitive impairment (MCI) is a translational state between cognitive changes of normal aging and AD. Lipid metabolism and pathogenesis of Alzheimer's disease (AD) are closely linked. Despite obviously discrete lipidome constitutions across lipoproteins, lipidomic approaches of AD has been mostly conducted without considering lipoprotein-dependent alterations. This study introduces a combination of asymmetrical flow field-flow fractionation (AF4) and nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry (nUHPLC-ESI-MS/MS) for a comprehensive lipid profiling in different lipoprotein level of patients plasma with AD and amnestic MCI in comparison to age-matched healthy controls. Lipoproteins in plasma samples were size-sorted by a semi-preparative scale AF4, followed by non-targeted lipid identification and high speed targeted quantitation with nUHPLC-ESI-MS/MS. It shows 14 significantly altered high abundance lipids in AD, exhibiting2-fold increases (p 0.01) in LDL/VLDL including triacylglycerol, ceramide, phosphatidylethanolamine, and diacylglycerol. Three lipid species (triacylglycerol 50:1, diacylglycerol 18:1_18:1, and phosphatidylethanolamine 36:2) showing a strong correlation with the degree of brain atrophy were found as candidate species which can be utilized to differentiate the early stage of MCI when simple Mini-Mental State Examination results were statistically incorporated. The present study elucidated lipoprotein-dependent alterations of lipids in progression of MCI and further to AD which can be utilized for the future development of lipid biomarkers to enhance the predictability of disease progress.

Rationale In addition to the development of adequate screening methods for multiple compounds, the World Anti-Doping Agency (WADA) requires anti-doping laboratories to analyze prohibited substances and their metabolites from various classes. This task presents a difficult challenge for all agencies and interests involved in the field of doping control. Methods A screening method is reported in which hybrid sample preparation was performed using a combination of weak cation-exchange solid-phase extraction (WCX-SPE) and the 'Dilute and Shoot' strategy in order to take advantage of both the methodologies. Target substances were extracted using a WCX cartridge and reconstituted with a diluted sample aliquot that included 20% of an untreated urine sample. The target substances were further analyzed by high-performance liquid chromatography/triple quadrupole mass spectrometry (LC/MS). Results The SPE procedure was optimized using a cartridge-washing step, elution conditions, and elution volume. The cartridge-washing step, which was performed using 10% methanol, improved the overall recovery of target substances. Since the recovery was observed to vary according to the pH of the eluting solution, we applied an elution step using both an acid and a basic organic solvent to achieve complementary recovery. Reconstitution of the diluted aliquot sample was performed to recover the polar substances. Conclusions The method was validated and applied to real samples in accordance with the external quality assessment scheme of WADA and to the previously reported samples that had provided positive test results. This novel method using hybrid sample preparation and LC/MS could be useful to screen multiple classes of the 264 targeted substances in anti-doping analysis.

In this Article, we have reported a fully automated online method to carry out proteolysis and glycopeptide enrichment in sequence for nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS) analysis. By implementing two serial thermoresponsive porous polymer membrane reactors (TPPMRs), in which the TPPM could be immobilized either with trypsin for proteolysis or with lectins for glycopeptide enrichment, the entire pretreatment procedure can be performed online in about an hour. The TPPM was fabricated by coating polystyrene-maleic anhydride- N-isopropylacrylamide (PS-MAn-PNIPAm), which was synthesized by reversible addition-fragmentation chain transfer polymerization, on a Nylon sheet. Because of the thermoresponsive nature of PNIPAm, it formed micelle cavities and changed its morphology at elevated temperatures, resulting in enhanced interactions between the enzyme or lectins and the proteins/peptides flowing through the membrane. The performances of the TPPMs were evaluated by varying the temperature conditions and the amount of standard proteins, showing that both proteolysis and glycopeptide enrichment with online deglycosylation were highly efficient at 37 °C. The developed online serial TPPMRs-nLC-ESI-MS/MS method was applied to the human plasma sample (1.5 μL) and a total of 262 N-glycopeptides could be identified from 155 glycoproteins. Thus, the present work demonstrates a fully automated high speed analytical protocol for online proteolysis and glycopeptide enrichment, which is extremely useful for analyzing small amounts of the proteome samples.

As lipidomics has attracted increased attention in life science, advanced mass spectrometry (MS) technologies have been combined with other separation techniques to improve and expand the branch of study. This review intends to provide general knowledge of offline and online coupling of flow field-flow fractionation (FlFFF)—a technique that encompasses the separation of nano- to micro-scale biomolecules—with MS for analysis of blood plasma lipoproteins, processes that are considered bottom-up and top-down approaches, respectively. The first part of this review focuses on the bottom-up method using multiplexed hollow fiber FlFFF (MxHF5) and nanoflow liquid chromatography electrospray-ionization tandem mass spectrometry (nLC-ESI–MS/MS) for non-targeted identification of lipids. In this protocol, plasma lipoproteins of different types are collected using MxHF5, and the lipids within the lipoproteins are then extracted and analyzed via nLC-ESI–MS/MS. The second part of the review describes the top-down approach, which uses online coupling of miniaturized FlFFF to ESI–MS for a fast screening of targeted lipids. Here, the separation of lipoproteins and detection of their component lipids are achieved simultaneously. While both methods aim to quantify the lipids within lipoproteins, the bottom-up approach provides an extensive lipidome, whereas the top-down method is suitable for high-speed targeted lipidomic analysis. This review discusses variants of FlFFF-ESI–MS/MS that offer effective analytical technologies for lipidomics.

In this study, ultrahigh-molecular-weight (MW) (>107Da) cationic polyacrylamides (C-PAMs), which are water-soluble polymers used in waste water treatment, were characterized using frit-inlet asymmetrical flow field-flow fractionation coupled with multi-angle light scattering and differential refractive detection. C-PAMs copolymerized with acryloxyethyltrimethyl ammonium chloride (DAC) were prepared by varying the feed amount of cationic monomer, polymerization method (solution vs. emulsion), and degree of branching. The MW of the copolymers prepared using emulsion polymerization (107-109Da) was generally larger than that of copolymers prepared using solution polymerization (4×107-108Da). When the amount of cationic monomer was increased from 10 to 55mol% in solution polymerization, hydrophobic contraction of the core induced formation of more compact C-PAMs. The copolymers prepared using emulsion polymerization formed highly aggregated or supercoil structures owing to increased intermolecular hydrophobic interaction when less cationic monomer was used. However, the MW decreased with increased cationic group content. In addition, C-PAMs larger than ∼108Da prepared using the emulsion method were separated by steric/hyperlayer elution mode while those in the 107-108Da range were analyzed in either normal or steric/hyperlayer mode depending on the decay patterns of field programming. Moreover, branched copolymers were found to be resolved with different elution modes under the same field decay pattern depending on the degree of branching: steric/hyperlayer for low-branching and normal for high-branching C-PAMs.

In this study, lipid analysis based on isotope-labeled methlylation (ILM) was performed by nanoflow ultrahigh performance liquid chromatography-eletrospray ionization-tandem mass spectrometry (nUPLC-ESI-MS/MS) for enhanced detection and quantification of targeted phospholipids. ILM depends on methylation of phosphate groups by (trimethylsilyl)diazomethane, and the ILM based quantitation with reversed phase nUPLC-ESI-MS/MS provides advantages in PL profiling such as enhanced detectability of methylated PLs owing to increased hydrophobicity and substantial increase in resolution due to the increase of retention. Efficacy of ILM in nUPLC-ESI-MS/MS analysis was evaluated in the selected reaction monitoring (SRM) method by varying the mixing ratio of H-/D-methylated PL standards, which resulted in the successful quantification of 24 species, including phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylglycerol (PG), ceramide-1-phosphate (Cer1P), phosphoinositides, and cardiolipin (CL), with ∼6.6% variation in the calculated ratio of H-/D-methylated PLs. The method was applied to the lipid extracts from a DU145 cell line after D-allose treatment, resulting in the quantification of 83 PLs of which results were not statistically different from those obtained by conventional quantification methods. Morever, detection and quantification of CLs and PAs were evidenced to be highly effective when used with the ILM method as 43 CLs and 20 PAs from cellular lipid extracts were analyzed while only 18 CLs and 12 PAs were identified when conventional methods were carried out. This proves the ILM combined with LC-MS to be a promising method for analysis of the aforementioned classes of lipids. Overall, the study highlighted the applicability of targeted quantification by the ILM method in lipidomic analysis and demonstrated an improvement in the detection of less abundant anionic PLs.

In this work, a unique D-amino acid oxidase reactor for enhanced enzymolysis efficiency is presented. A kind of magnetic polymer matrices, composed of iron oxide nanoparticles and porous polymer membrane (poly styrene-co-maleic anhydride), was prepared. With covalent bonding D-Amino acid oxidase on the surface of the matrices and characterization of scanning electron microscope and vibrating sample magnetometer, it demonstrated that the membrane enzyme reactor was successfully constructed. The enzymolysis efficiency of the enzyme reactor was evaluated and the apparent Michaelis-Menten constants of D-Amino acid oxidase were determined (Km was 1.10mM, Vmax was 23.8mMmin-1) by a chiral ligand exchange capillary electrophoresis protocol with methionine as the substrate. The results indicated that the enzyme reactor could exhibit good stability and excellent reusability. Importantly, because the enzyme and the substrate could be confined into the pores of the matrices, the enzyme reactor displayed the improved enzymolysis efficiency due to the confinement effect. Further, the prepared enzyme reactor was applied for D-Amino acid oxidase inhibitors screening. It has displayed that the proposed protocol could pave a new way for fabrication of novel porous polymer membrane based enzyme reactors to screen enzyme inhibitors.

This study aimed to measure phytoestrogen contents in health functional foods (HFFs) to alleviate menopausal symptoms that are commercially available in South Korea. A sensitive and selective method using gas chromatography–mass spectrometry (GC-MS) for the monitoring of 21 phytoestrogens was developed and fully validated. Phytoestrogens were extracted by liquid–liquid extraction using methyl tert-butylether as the solvent. Food samples were enzymatically hydrolyzed with β-glucuronidase/arylsulfatase. Before GC-MS, the analytes were derivatized to their trimethylsilyl derivatives by incubation with N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane for 30 min at 80°C. The calibration curves showed good linearity (r 2 > 0.99) in the range of interest for each analyte. The limits of detection and quantification of the 21 phytoestrogens were in the ranges of 0.5–20 and 1–50 ng/mL, respectively. The intra- and inter-day precisions were all below 15.7%. These multivariate procedures were efficient in determining the optimal conditions using peak heights as responses.

Exosomes are membrane-bound extracellular vesicles involved in intercellular communication and tumor cell metastasis. In this study, flow field-flow fractionation (FlFFF) was utilized to separate urinary exosomes by size, demonstrating a significant difference in exosome sizes between healthy controls and patients with prostate cancer (PCa). Exosome fractions of different sizes were collected for microscopic analysis during an FlFFF run and evaluated with exosome marker proteins using Western blot analysis. The results indicated that exosomes of different sizes originated from different types of cells. Collected exosome fractions were further examined using nanoflow ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (nUPLC-ESI-MS/MS) for lipidomic analysis. A total of 162 lipids (from 286 identified) were quantified using a selected reaction monitoring (SRM) method. The overall amount of lipids increased by 1.5- to 2-fold in patients with PCa and degree of increase was more significant in the smaller fractions (diameter150 nm) than in the larger ones (diameter150 nm) some classes of lipids. In addition, neutral lipids like diacylglycerol (DAG) and triacylglycerol (TAG) decreased in all exosomes without size dependency. Moreover, a dramatic increase in 22:6/22:6-phosphatidylglycerol (PG) was observed and significant decrease in (16:0,16:0)- and (16:1, 18:1)-DAG species (nearly 5-fold) and high abundant TAG species (2.5-fold) was observed in patients with PCa. The results of this study indicate that FlFFF can be employed for the high-speed screening of urinary exosome sizes in patients with PCa and lipidomic analysis of the fractionated exosomes has potential for developing and distinguishing biomarkers of PCa.

Acidic lipids are associated with the regulation of the structure and function of membrane proteins. Therefore, accurate and highly precise analysis of acidic lipids is important for elucidating their biological roles and pathological mechanisms. In this study, an enhanced analytical method for the separation and quantification of acidic lipids, including phosphatidylserine (PS), phosphatidic acid (PA), cardiolipin, and their lyso-derivatives, was developed using nanoflow ultrahigh performance liquid chromatography-electrospray ionisation-tandem mass spectrometry. The separation and mass spectrometry detection of acidic lipids were optimised in terms of peak tailing and time-based separation efficiencies, with carbamate-embedded C18 as the stationary phase, in the presence of an appropriate liquid chromatography solvent modifier. This newly developed method was applied to analyse a lipid extract from porcine brain. A significant increase in the number of acidic lipids identified (176 vs. 134), including intact monolysocardiolipin (17 vs. 4), was observed with the new method compared with conventional C18. The quantification of acidic lipids was validated with plasma standard (NIST SRM 1950) spiked with a number of LPS and PS standards, and acceptable accuracy (15%) was obtained. The present method was found to be reliable for the acidic lipid analysis based on qualitative results from tissue extract and plasma samples.

Lipids are important signaling molecules regulating biological processes under normal and diseased conditions. Although p53 mutation is well-known for causing cancer, the relationship between p53-related tumorigenesis and altered lipid profile is unclear. We profiled differences in lipid expressions in liver, lung, and kidney in p53 knockout (KO) mice by high-speed quantitative analysis of 320 lipids (399 species identified) using nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry (nUPLC-MS/MS). Lung tissues were most severely affected by the lack of p53 gene, as shown by significant reduction (24-44%, P0.05) in total phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SM), diacylglycerol (DG), and triacylglycerol (TG), and significant increases (30-50%) in phosphatidylserine (PS), phosphatidylinositol (PI), and monohexosylceramide (MHC). MHC levels increased in all tissues. Dihexosylceramide (DHC) level decreased only in kidney tissue. Most PI, PS, and phosphatidic acid (PA) species showing significant increases contained a saturated acyl chain (18:0) in lung and liver tissues. Neutral glycerolipids (16:0/22:0-DG and most TGs with saturated and monounsaturated acyl chains) decreased 2-4-fold in the liver tissue. Our results suggest that the lack of p53 and altered lipid profiles are closely related, but as their changes vary from one tissue to another, the lipid alterations are tissue-specific.

An on-line lipid extraction method is introduced by utilizing a short capillary extraction column using HILIC and C4 particles prior to nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS). The on-line extraction using a urine sample spiked with PL standards showed similar or slightly higher recovery values (86%-96%) of phospholipids (PLs) compared to those obtained by the conventional off-line extraction based on the Folch method with or without using the air-exposed drying process. In this study, we demonstrated that PL oxidation can occur during the air-exposed drying process of lipid extracts in standard liquid-liquid extraction procedures, which was confirmed by the oxidized PL (OxPL) molecules that were generated from an off-line extraction using a few PL standards. Quantitative comparison of these OxPL species between on- and off-line extraction followed by nLC-MS/MS with multiple reaction monitoring (MRM) analysis showed a significant decrease (2-10 fold) in unwanted OxPL species when on-line extraction was employed. While the number of identified PLs from a urine sample was somewhat lower than those by off-line extraction, the number of OxPLs was significantly reduced (from 70 to 22) with on-line extraction. The new method offers high speed (∼5min) automated extraction of PLs with nLC-MS/MS analysis and presents the possibility of handling a biological sample with a very limited amount of lipids.

A rapid and high-throughput quantification method (approximately 300 lipids within 20 min) was established using nanoflow ultrahigh-pressure liquid chromatography-tandem mass spectrometry (nUPLC-ESI-MS/MS) with selective reaction monitoring (SRM) and applied to the quantitative profiling of the hepatic lipids of rabbits with different metabolic conditions that stimulate the development of nonalcoholic fatty liver disease (NAFLD). Among the metabolic conditions of rabbits in this study [inflammation (I), high-cholesterol diet (HC), and high-cholesterol diet combined with inflammation (HCI)], significant perturbation in hepatic lipidome (3-fold and p 0.01) was observed in the HC and HCI groups, while no single lipid showed a significant change in group I. In addition, this study revealed a dramatic increase (2-fold) in relatively high-abundant monohexosylceramides (MHCs), sphingomyelins (SMs), and triacylglycerols (TGs) in both the HC and HCI groups, especially in MHCs as all 11 MHCs increased by larger than 3- to 12-fold. As the levels of the relatively high-abundant lipids in the above classes increased, the total lipidome level of each class increased significantly by approximately 2-fold to 5-fold. Other classes of lipids also generally increased, which was likely induced by the increase in mitogenic and nonapoptotic MHCs and SMs, as they promote cell proliferation. On the other hand, a slight decrease in the level of apoptotic ceramides (Cers) was observed, which agreed with the general increase in total lipid level. As distinct changes in hepatic lipidome were observed from HC groups, this suggests that HC or HCI is highly associated with NAFLD but not inflammation alone itself. Graphical Abstract Schematic of lipidomic analysis from hepatic tissue using nanoflow LC-ESI-MS/MS and nUPLC-ESI-MS/MS.

A deficiency of α-galactosidase A causes Fabry disease (FD) by disrupting lipid metabolism, especially trihexosylceramide (THC). Enzyme replacement therapy (ERT) is clinically offered to FD patients in an attempt to lower the accumulated lipids. Studies on specific types of lipids that are directly or indirectly altered by FD are very scarce, even though they are crucial in understanding the biological process linked to the pathogenesis of FD. We performed a comprehensive lipid profiling of plasma and urinary lipids from FD patients with nanoflow liquid chromatography electrospray-ionization tandem mass spectrometry (nLC-ESI-MS/MS) and identified 129 plasma and 111 urinary lipids. Among these, lipids that exhibited alternations (>twofold) in patients were selected as targets for selected reaction monitoring (SRM)-based high-speed quantitation using nanoflow ultra-performance LC-ESI-MS/MS (nUPLC-ESI-MS/MS) and 31 plasma and 26 urinary lipids showed significant elevation among FD patients. Higher percentages of sphingolipids (SLs; 48% for plasma and 42% for urine) were highly elevated in patients; whereas, a smaller percentage of phospholipids (PLs; 15% for plasma and 13% for urine) were significantly affected. Even though α-galactosidase A is reported to affect THC only, the results show that other classes of lipids (especially SLs) are changed as well, indicating that FD not only alters metabolism of THC but various classes of lipids too. Most lipids showing significant increases in relative amounts before ERT decreased after ERT, but overall, ERT influenced plasma lipids more than urinary lipids.

Fast and effective protein digestion is a vital process for mass spectrometry (MS) based protein analysis. This study introduces a porous polymer membrane enzyme reactor (PPMER) coupled to nanoflow liquid chromatography-tandem MS (nLC-ESI-MS/MS) for on-line digestion and analysis of proteins. Poly (styrene-co-maleic anhydride) (PS-co-MAn) was fabricated by the breath figure method to make a porous polymer membrane in which the MAn group was covalently bound to enzyme. Based on this strategy, microscale PPMER (μPPMER) was constructed for on-line connection with the nLC-ESI-MS/MS system. Its capability for enzymatic digestion with bovine serum albumin (BSA) was evaluated with varied digestion periods. The on-line proteolysis of BSA and subsequent analysis with μPPMER-nLC-ESI-MS/MS revealed that peptide sequence coverage increased from 10.3% (digestion time 10 min) to 89.1% (digestion time 30 min). μPPMER can efficiently digest proteins due to the microscopic confinement effect, showing its potential application in fast protein identification and protease immobilization. Applications of on-line digestion using μPPMER with human plasma and urinary proteome samples showed that the developed on-line method yielded equivalent or better performance in protein coverage and identified more membrane proteins than the in-solution method. This may be due to easy accommodation of hydrophobic membrane proteins within membrane pores.

In this study, frit inlet asymmetrical flow field-flow fractionation (FlFFF) with multi-angle light scattering (MALS) and differential refractive index (DRI) detection is utilized for size separation, determination of molecular weight (MW), and conformation of ultrahigh-MW (10(7)-10(9) g/mol) cationic polyacrylamides (C-PAMs), a class of water-soluble copolymers based on acrylamide and vinyl-type comonomers with quaternary ammonium cations that are widely used in wastewater treatment and in paper industries. Linear and branched C-PAM copolymers prepared in two different polymerization methods (solution and emulsion) from varying amounts of crosslinking agent and initiator were size fractionated by FlFFF with field-programming. It was found experimentally that the linear copolymers from both polymerization methods were less than 10(8) g/mol in MW with compact, nearly spherical structures, while the branched C-PAM copolymers from the emulsion polymerization showed a significant increase in average MW up to ∼ 10(9)g/mol, which was about 20-fold greater than those from the solution method, and the branched copolymers had more compact or shrunken conformations. While both linear and branched copolymers less than 10(8) g/mol MW were well resolved in an increasing order of MW (normal mode), it was noted that branched copolymers prepared through emulsion polymerization exhibited significantly larger MWs of 10(8-)10(9) g/mol and eluted in the steric/hyperlayer mode, in which the elution order is reversed in an extreme run condition (strong initial field strength followed by a fast field decay during programming).