This document presents a targeted liquid chromatography-mass spectrometry workflow for comprehensive profiling of sphingolipids. The workflow improves upon previous methods by using (1) an optimized extraction procedure, (2) a segmented gradient to better separate isobaric species, and (3) parallel reaction monitoring of sphingolipid fragments to identify species. The workflow was tested on RAW 264.7 cells and quantified 61 sphingolipids over a wide dynamic range with low femtomole detection limits, making it a high-throughput tool for sphingolipidomics.
This document describes the synthesis and characterization of a series of anionic liposaccharide derivatives intended to act as absorption enhancers and improve oral bioavailability of drugs. The liposaccharides were designed with a lipophilic lipid side chain and a hydrophilic head containing glucose and glutamic acid. Isothermal titration calorimetry was used to determine the critical aggregation concentrations and thermodynamic profiles of the liposaccharides. Two liposaccharides formed nanoparticles below 100 nm and had critical aggregation concentrations below 0.325 mM, indicating favorable aggregation in aqueous solution driven by entropy.
Effectiveness Of The Treatment Before And After The...Olga Bautista
The document discusses cation exchange gel samples labeled IEX 19-22 that had the highest activity and were selected for analysis by gel electrophoresis. The gel banding patterns were the same across samples, allowing them to be combined for yield calculations. While dye ligand chromatography resulted in a lower purification than expected, it concentrated the protein of interest compared to earlier fractions as determined by activity assays.
The arrangement of the large (70,000 Mr) and small (30,000 Mr) subunits of succinate dehydrogenase (SDH) in the mitochondrial inner membrane was investigated using limited proteolysis and immunoblotting. Both subunits were resistant to proteinase treatment when the inner membrane integrity was preserved, suggesting neither subunit is exposed at the cytoplasmic surface. The small subunit appears to protrude into the matrix compartment, as it is extensively degraded but no membrane-associated fragment is observed. The large subunit interacts with the matrix side via two distinct domains that are detected as stable membrane-associated fragments after proteinase treatment, though one domain can be further degraded. This suggests the large subunit membrane interaction occurs via two regions,
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research...iosrphr_editor
This document summarizes research on the formulation, optimization, and characterization of atorvastatin-loaded solid lipid nanoparticles (SLNs). Atorvastatin-loaded SLNs were prepared using the hot homogenization followed by ultrasonication method. The effects of lipid composition, surfactant mixture, and sonication time on particle size, polydispersity index, zeta potential, drug entrapment efficiency, and in vitro drug release were investigated. The optimized formulation had a mean particle size of 50.0±6.12 nm, polydispersity index of 0.08±0.011, zeta potential of 10.40±4.68mV, and drug entrapment efficiency of 88.7
This study evaluates an internal standard cocktail developed for the LIPID MAPS Consortium for quantitative analysis of sphingolipids using mass spectrometry. The internal standard cocktail contains uncommon chain length analogs of sphingoid bases and sphingolipids that have similar ionization and fragmentation properties to the target analytes. Two mass spectrometers, a triple quadrupole and a quadrupole linear ion trap, were used to optimize the method and validate the internal standard cocktail. The internal standard cocktail and mass spectrometry methods were applied to analyze sphingolipids extracted from RAW264.7 mouse macrophage cells.
The document describes the synthesis and evaluation of nitromethane substituted chalcone derivatives for their antimicrobial and antitubercular activity. Chalcones were synthesized from substituted acetophenones and aldehydes under basic conditions. Michael adducts were obtained by reacting the chalcones with nitromethane under Michael addition reaction conditions. The structures of the compounds were characterized using NMR and IR spectroscopy. Docking studies were performed to study the binding interactions of the compounds with various Mycobacterium tuberculosis protein targets. The compounds were evaluated for their in vitro antitubercular, anthelmintic and antimicrobial activity. Some compounds showed good activity against Mycobacterium tuberculosis and against various microorgan
This document describes research into synthesizing and evaluating Michael adducts derived from chalcones for biological activity. Michael adducts were synthesized by reacting chalcones with nitromethane under basic conditions. Various chalcones were first synthesized by reacting substituted acetophenones with substituted aldehydes under basic conditions. The resulting Michael adducts underwent molecular docking studies against various Mycobacterium tuberculosis protein targets as well as evaluation for anti-tubercular, anthelmintic, and antimicrobial activity. Several of the synthesized compounds showed promising anti-tubercular activity at 50μg/ml and good anthelmintic activity, suggesting electron donating groups aid activity.
This document summarizes a proteomic analysis of the excretory/secretory (ES) proteins of Toxoplasma gondii, the causative agent of toxoplasmosis. 34 proteins were identified from the ES proteins of the RH strain of T. gondii using LC/MS/MS analysis and spectral counting methods. The three most abundant proteins identified were GRA1, GRA7, and GRA2. A variety of other proteins were also identified, including micronemal proteins, rhoptry proteins, and dense granule proteins, which play important roles in T. gondii host cell invasion and survival within host cells. This analysis provides new insights into the ES proteome of T
This document describes the synthesis and characterization of a series of anionic liposaccharide derivatives intended to act as absorption enhancers and improve oral bioavailability of drugs. The liposaccharides were designed with a lipophilic lipid side chain and a hydrophilic head containing glucose and glutamic acid. Isothermal titration calorimetry was used to determine the critical aggregation concentrations and thermodynamic profiles of the liposaccharides. Two liposaccharides formed nanoparticles below 100 nm and had critical aggregation concentrations below 0.325 mM, indicating favorable aggregation in aqueous solution driven by entropy.
Effectiveness Of The Treatment Before And After The...Olga Bautista
The document discusses cation exchange gel samples labeled IEX 19-22 that had the highest activity and were selected for analysis by gel electrophoresis. The gel banding patterns were the same across samples, allowing them to be combined for yield calculations. While dye ligand chromatography resulted in a lower purification than expected, it concentrated the protein of interest compared to earlier fractions as determined by activity assays.
The arrangement of the large (70,000 Mr) and small (30,000 Mr) subunits of succinate dehydrogenase (SDH) in the mitochondrial inner membrane was investigated using limited proteolysis and immunoblotting. Both subunits were resistant to proteinase treatment when the inner membrane integrity was preserved, suggesting neither subunit is exposed at the cytoplasmic surface. The small subunit appears to protrude into the matrix compartment, as it is extensively degraded but no membrane-associated fragment is observed. The large subunit interacts with the matrix side via two distinct domains that are detected as stable membrane-associated fragments after proteinase treatment, though one domain can be further degraded. This suggests the large subunit membrane interaction occurs via two regions,
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research...iosrphr_editor
This document summarizes research on the formulation, optimization, and characterization of atorvastatin-loaded solid lipid nanoparticles (SLNs). Atorvastatin-loaded SLNs were prepared using the hot homogenization followed by ultrasonication method. The effects of lipid composition, surfactant mixture, and sonication time on particle size, polydispersity index, zeta potential, drug entrapment efficiency, and in vitro drug release were investigated. The optimized formulation had a mean particle size of 50.0±6.12 nm, polydispersity index of 0.08±0.011, zeta potential of 10.40±4.68mV, and drug entrapment efficiency of 88.7
This study evaluates an internal standard cocktail developed for the LIPID MAPS Consortium for quantitative analysis of sphingolipids using mass spectrometry. The internal standard cocktail contains uncommon chain length analogs of sphingoid bases and sphingolipids that have similar ionization and fragmentation properties to the target analytes. Two mass spectrometers, a triple quadrupole and a quadrupole linear ion trap, were used to optimize the method and validate the internal standard cocktail. The internal standard cocktail and mass spectrometry methods were applied to analyze sphingolipids extracted from RAW264.7 mouse macrophage cells.
The document describes the synthesis and evaluation of nitromethane substituted chalcone derivatives for their antimicrobial and antitubercular activity. Chalcones were synthesized from substituted acetophenones and aldehydes under basic conditions. Michael adducts were obtained by reacting the chalcones with nitromethane under Michael addition reaction conditions. The structures of the compounds were characterized using NMR and IR spectroscopy. Docking studies were performed to study the binding interactions of the compounds with various Mycobacterium tuberculosis protein targets. The compounds were evaluated for their in vitro antitubercular, anthelmintic and antimicrobial activity. Some compounds showed good activity against Mycobacterium tuberculosis and against various microorgan
This document describes research into synthesizing and evaluating Michael adducts derived from chalcones for biological activity. Michael adducts were synthesized by reacting chalcones with nitromethane under basic conditions. Various chalcones were first synthesized by reacting substituted acetophenones with substituted aldehydes under basic conditions. The resulting Michael adducts underwent molecular docking studies against various Mycobacterium tuberculosis protein targets as well as evaluation for anti-tubercular, anthelmintic, and antimicrobial activity. Several of the synthesized compounds showed promising anti-tubercular activity at 50μg/ml and good anthelmintic activity, suggesting electron donating groups aid activity.
This document summarizes a proteomic analysis of the excretory/secretory (ES) proteins of Toxoplasma gondii, the causative agent of toxoplasmosis. 34 proteins were identified from the ES proteins of the RH strain of T. gondii using LC/MS/MS analysis and spectral counting methods. The three most abundant proteins identified were GRA1, GRA7, and GRA2. A variety of other proteins were also identified, including micronemal proteins, rhoptry proteins, and dense granule proteins, which play important roles in T. gondii host cell invasion and survival within host cells. This analysis provides new insights into the ES proteome of T
This document describes research on novel ultra-short peptide sequences containing tryptophan and arginine residues that show potent antibacterial activity against drug-resistant Staphylococcus aureus. Key findings include:
1) The peptide sequences were modified with N-terminal aromatic tags to induce self-assembly into nanovesicles around 140-190 nm in diameter.
2) The nanovesicles have a positively charged surface and hydrophobic core containing tryptophan residues and N-terminal tags.
3) Experiments showed the peptide nanovesicles kill bacteria by disrupting their membranes, causing membrane depolarization and leakage of intracellular contents.
4) The self-assembled nanostructure is believed to
This document is a letter informing the author that changes made to the HTML version of their article will be added before publication but are not reflected in the attached PDF file. The letter also notes that the PDF should not be used for submitting corrections. The PDF then contains a research article examining the structure-activity relationships of redox active thiol peroxidase mimic compounds. The study relates the ability of organoselenium and organotellurium compounds to catalyze the oxidation of glutathione and dihydrolipoic acid to their cytotoxicity and ability to act as antioxidants or prooxidants in cancer cells. The results show dihydrolipoic acid oxidation correlates with cytotoxicity and prooxidant action, allowing prediction of compound
This document discusses materials and methods used in a study involving the chemical fipronil and zinc. Twenty male albino rats were divided into four groups of five rats each: a control group, a zinc group that received zinc supplementation, a fipronil group exposed to the insecticide fipronil, and a combination group exposed to both zinc and fipronil. Biochemical assays were conducted to assess oxidative stress markers like superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, glutathione, lipid peroxidation, and total protein in the rats. Chemicals used including fipronil and zinc sulfate were obtained from reputable suppliers. Kits for the biochemical assays were purchased from a diagnostic
Bayer et al. - 2012 - Purification and characterization of riproximin frCristina Voss
This document describes the purification and characterization of riproximin, a type II ribosome inactivating protein, from the fruit kernels of Ximenia americana. The researchers established a purification procedure involving aqueous extraction of the kernels, removal of lipids using chloroform, and chromatographic purification steps on an anion exchange resin and lactosyl-Sepharose. The purified riproximin from fruit kernels was biochemically and biologically similar to riproximin from the original African plant material, but differences were found in electrophoresis patterns and mass spectrometry profiles, suggesting the purified version contains closely related isoforms. The reliable purification process provides a basis for further research on this potential anticancer drug candidate.
Antimicrobial agents and chemotherapy 2005 dartoisSinkope
This document describes research into novel synthetic cyclic peptides with antibacterial activity. Six peptides were selected based on in vitro potency for further study. These peptides showed efficacy in mouse models of peritonitis and thigh infection caused by methicillin-sensitive and methicillin-resistant Staphylococcus aureus at doses of 4.0-8.0 mg/kg. The pharmacokinetics of the peptides in mice were determined, with compounds showing poorer efficacy having lower serum concentrations and higher volumes of distribution. S. aureus did not easily develop resistance to the peptides. The cyclic peptides show potential as systemic antibacterial agents for resistant infections.
This document discusses tracing the evolution of the human body through analyzing the chemical evolution of proteins and other molecules in the body over time. It notes that tracking changes in conserved proteins that control fundamental processes, like the Pax6 gene which regulates eye development, can reveal how closely related different organisms are. It recommends using the human arrestin protein sequence as an example, performing BLAST searches to find the arrestin sequence in the human genome and other organism genomes, then aligning the protein sequences to compare percentages and determine evolutionary relationships.
This document describes computational techniques used to design novel competitive inhibitors of the E. coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTN) enzyme. It utilized core hopping to generate 10,000 structures by varying the core while keeping functional groups constant. Docking and binding energies were calculated for subsets of compounds down to the top 8 ligands. Results show several compounds have more favorable predicted binding than the control TDI inhibitor, warranting further optimization and testing of lead compounds.
1) The document describes a novel method called INLIGHTTM for the relative quantification of N-linked glycans using isotopically labeled glycan hydrazide tags.
2) The method involves releasing N-linked glycans from glycoproteins using PNGase F, then derivatizing the glycans from two samples with either a light or heavy tagged reagent.
3) The tagged glycans are mixed, analyzed by LC/MS, and ratios of light and heavy glycan pairs are calculated to quantify differences between the two samples after correcting for isotopic overlap.
This study investigated the binding targets of the inhaled anesthetic halothane in rat brain. The researchers used quantitative photoaffinity labeling and electrophoresis to analyze rat cerebellar homogenates exposed to [14C]halothane. They found that halothane incorporated into many soluble and membrane-bound proteins, with stoichiometry values ranging from 0 to 4 and apparent IC50 values from 0.2 to 2.0 mM. Competition experiments showed that unlabeled halothane, chloroform, and isoflurane inhibited halothane labeling to varying degrees, while a non-anesthetic compound inhibited the least. These results suggest that halothane binding motifs can be found in a wide variety of proteins in the brain
This document describes the design, synthesis, and evaluation of novel thrombin inhibitors incorporating P3-P4 lactam sulfonamide moieties. The inhibitors were designed to exploit interactions with thrombin's S2 and S3 sites to improve selectivity over related serine proteases. A series of 5-7 membered lactam rings were synthesized and incorporated a P1 argininal group. X-ray crystallography of one inhibitor bound to thrombin confirmed the predicted binding mode. In vitro testing showed several inhibitors had low nanomolar IC50 values against thrombin and good selectivity over trypsin and factor Xa. Overall, the lactam sulfonamides represent a new class of orally bioavailable
This study investigated the structural, thermodynamic and unfolding properties of the kappa class glutathione transferase (CdGSTK1-1) from Camelus dromedarius. The key findings were:
1) CdGSTK1-1 was expressed in E. coli and purified. Analytical gel filtration showed it has a unique trimeric structure.
2) CdGSTK1-1 exhibited low thermal stability and unfolded through three states with intermediate species formation. The first transition melting point was 40.3°C and the second was 49.1°C.
3) Intrinsic fluorescence and near-UV CD studies indicated that substrate binding did not cause major conformational changes in
This document summarizes a study examining the anti-proliferative effects of Arisaema intermedium lectin (AIL) on human colon cancer cells. AIL was purified from Arisaema intermedium tubers and its effects were tested on HCT-15 colon cancer cells. MTT assays showed AIL inhibited the growth of HCT-15 cells in a dose-dependent manner, with 53% inhibition at 100 μg/mL AIL. Further experiments found AIL treatment led to DNA fragmentation, changes in nucleic acid content, cell morphology changes, and decreased cell viability, indicating AIL induces apoptosis in HCT-15 cells. The results suggest AIL has anti-cancer properties and works through an apoptotic
The document describes the design, synthesis, and testing of novel lipopeptide conjugates as potential antimicrobial agents. A library of conjugates was designed based on a template consisting of a hydrophobic moiety, dipeptide, and spermidine. Conjugates containing linoleic acid exhibited potent antibacterial activity against both Gram-positive and Gram-negative bacteria. Structure-activity relationships revealed that optimal activity required a hydrophobicity of 50-70% and a minimum charge of +2. Active conjugates were found to have different modes of action, damaging bacterial cell membranes and DNA. Two conjugates showed selective membrane disruption of pathogenic MRSA and were identified as promising leads for further optimization and development as antimicrobial agents.
Liposomal drug delivery involves encapsulating drugs within liposomes, which are spherical vesicles composed of phospholipid bilayers, to improve drug targeting and reduce toxicity. Liposomes can be classified based on lamellarity, size, and method of preparation. Drugs are encapsulated within the aqueous interior or phospholipid bilayer of liposomes. Liposomes protect drugs, control drug release, and can be targeted to specific tissues. Applications include cancer therapy, antimicrobial delivery, ophthalmic delivery, and topical delivery to improve treatment.
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online
Crimson Publishers-Stable Labeled Isotopes as Internal Standards: A Critical ...CrimsonPublishersMAPP
SIL internal standards are commonly used in LC-MS/MS analyses to improve accuracy and precision. There are two main types: structurally similar analogs or isotopically labeled compounds containing stable isotopes like deuterium, carbon-13, or nitrogen-15. SIL standards provide structural information to understand analyte fragmentation patterns and metabolism. Their use has been shown to reduce ionization variations compared to analog standards. However, SIL standards can still cause ion suppression or enhancement and may not fully correct for matrix effects. While very useful, SIL standards also have limitations like expense and potential for different retention times versus analytes. Overall they remain a preferred choice but alternative standards may still be needed in some cases.
1) The study examined the effects of various cytokinins, cytokinin ribosides, and their analogs on the viability of normal and cancerous human cells.
2) Only a few compounds, including isopentenyladenosine (1a) and N6-benzyladenosine (3a), significantly impaired viability in most cell lines tested, including normal endothelial cells and various cancer cell lines.
3) Colon carcinoma LoVo cells were uniquely insensitive to all compounds tested and may serve as a model for studying cytokinin resistance.
Recent Advancement and Patents of the Lipid Polymer Hybrid Nanoparticlespeertechzpublication
In recent years, robustness and surface engineering of dosage form made improvement in
pharmacokinetics with decrease in dose of drug. Specifi city with adherence of ligands has now become
the reality as surface modifi cation can easily deceive phagocytic system. Lipid molecules ensures the
release of drug at lymphatic system, entrapment of polymeric nanoparticles in lipoidal core led to the
avoidance of disadvantage of low entrapment effi ciency if use of hydrophobic drug with hydrophobic
polymer becomes essential. Various studies have been published and the best formulations with optimal
In vitro and In vivo results are highlighted in this paper. In this review most advanced researches and
accepted patents were discussed so to act as a medium for getting everything regarding lipid polymer
hybrid particles under one umbrella.
This document describes research on novel ultra-short peptide sequences containing tryptophan and arginine residues that show potent antibacterial activity against drug-resistant Staphylococcus aureus. Key findings include:
1) The peptide sequences were modified with N-terminal aromatic tags to induce self-assembly into nanovesicles around 140-190 nm in diameter.
2) The nanovesicles have a positively charged surface and hydrophobic core containing tryptophan residues and N-terminal tags.
3) Experiments showed the peptide nanovesicles kill bacteria by disrupting their membranes, causing membrane depolarization and leakage of intracellular contents.
4) The self-assembled nanostructure is believed to
This document is a letter informing the author that changes made to the HTML version of their article will be added before publication but are not reflected in the attached PDF file. The letter also notes that the PDF should not be used for submitting corrections. The PDF then contains a research article examining the structure-activity relationships of redox active thiol peroxidase mimic compounds. The study relates the ability of organoselenium and organotellurium compounds to catalyze the oxidation of glutathione and dihydrolipoic acid to their cytotoxicity and ability to act as antioxidants or prooxidants in cancer cells. The results show dihydrolipoic acid oxidation correlates with cytotoxicity and prooxidant action, allowing prediction of compound
This document discusses materials and methods used in a study involving the chemical fipronil and zinc. Twenty male albino rats were divided into four groups of five rats each: a control group, a zinc group that received zinc supplementation, a fipronil group exposed to the insecticide fipronil, and a combination group exposed to both zinc and fipronil. Biochemical assays were conducted to assess oxidative stress markers like superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, glutathione, lipid peroxidation, and total protein in the rats. Chemicals used including fipronil and zinc sulfate were obtained from reputable suppliers. Kits for the biochemical assays were purchased from a diagnostic
Bayer et al. - 2012 - Purification and characterization of riproximin frCristina Voss
This document describes the purification and characterization of riproximin, a type II ribosome inactivating protein, from the fruit kernels of Ximenia americana. The researchers established a purification procedure involving aqueous extraction of the kernels, removal of lipids using chloroform, and chromatographic purification steps on an anion exchange resin and lactosyl-Sepharose. The purified riproximin from fruit kernels was biochemically and biologically similar to riproximin from the original African plant material, but differences were found in electrophoresis patterns and mass spectrometry profiles, suggesting the purified version contains closely related isoforms. The reliable purification process provides a basis for further research on this potential anticancer drug candidate.
Antimicrobial agents and chemotherapy 2005 dartoisSinkope
This document describes research into novel synthetic cyclic peptides with antibacterial activity. Six peptides were selected based on in vitro potency for further study. These peptides showed efficacy in mouse models of peritonitis and thigh infection caused by methicillin-sensitive and methicillin-resistant Staphylococcus aureus at doses of 4.0-8.0 mg/kg. The pharmacokinetics of the peptides in mice were determined, with compounds showing poorer efficacy having lower serum concentrations and higher volumes of distribution. S. aureus did not easily develop resistance to the peptides. The cyclic peptides show potential as systemic antibacterial agents for resistant infections.
This document discusses tracing the evolution of the human body through analyzing the chemical evolution of proteins and other molecules in the body over time. It notes that tracking changes in conserved proteins that control fundamental processes, like the Pax6 gene which regulates eye development, can reveal how closely related different organisms are. It recommends using the human arrestin protein sequence as an example, performing BLAST searches to find the arrestin sequence in the human genome and other organism genomes, then aligning the protein sequences to compare percentages and determine evolutionary relationships.
This document describes computational techniques used to design novel competitive inhibitors of the E. coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTN) enzyme. It utilized core hopping to generate 10,000 structures by varying the core while keeping functional groups constant. Docking and binding energies were calculated for subsets of compounds down to the top 8 ligands. Results show several compounds have more favorable predicted binding than the control TDI inhibitor, warranting further optimization and testing of lead compounds.
1) The document describes a novel method called INLIGHTTM for the relative quantification of N-linked glycans using isotopically labeled glycan hydrazide tags.
2) The method involves releasing N-linked glycans from glycoproteins using PNGase F, then derivatizing the glycans from two samples with either a light or heavy tagged reagent.
3) The tagged glycans are mixed, analyzed by LC/MS, and ratios of light and heavy glycan pairs are calculated to quantify differences between the two samples after correcting for isotopic overlap.
This study investigated the binding targets of the inhaled anesthetic halothane in rat brain. The researchers used quantitative photoaffinity labeling and electrophoresis to analyze rat cerebellar homogenates exposed to [14C]halothane. They found that halothane incorporated into many soluble and membrane-bound proteins, with stoichiometry values ranging from 0 to 4 and apparent IC50 values from 0.2 to 2.0 mM. Competition experiments showed that unlabeled halothane, chloroform, and isoflurane inhibited halothane labeling to varying degrees, while a non-anesthetic compound inhibited the least. These results suggest that halothane binding motifs can be found in a wide variety of proteins in the brain
This document describes the design, synthesis, and evaluation of novel thrombin inhibitors incorporating P3-P4 lactam sulfonamide moieties. The inhibitors were designed to exploit interactions with thrombin's S2 and S3 sites to improve selectivity over related serine proteases. A series of 5-7 membered lactam rings were synthesized and incorporated a P1 argininal group. X-ray crystallography of one inhibitor bound to thrombin confirmed the predicted binding mode. In vitro testing showed several inhibitors had low nanomolar IC50 values against thrombin and good selectivity over trypsin and factor Xa. Overall, the lactam sulfonamides represent a new class of orally bioavailable
This study investigated the structural, thermodynamic and unfolding properties of the kappa class glutathione transferase (CdGSTK1-1) from Camelus dromedarius. The key findings were:
1) CdGSTK1-1 was expressed in E. coli and purified. Analytical gel filtration showed it has a unique trimeric structure.
2) CdGSTK1-1 exhibited low thermal stability and unfolded through three states with intermediate species formation. The first transition melting point was 40.3°C and the second was 49.1°C.
3) Intrinsic fluorescence and near-UV CD studies indicated that substrate binding did not cause major conformational changes in
This document summarizes a study examining the anti-proliferative effects of Arisaema intermedium lectin (AIL) on human colon cancer cells. AIL was purified from Arisaema intermedium tubers and its effects were tested on HCT-15 colon cancer cells. MTT assays showed AIL inhibited the growth of HCT-15 cells in a dose-dependent manner, with 53% inhibition at 100 μg/mL AIL. Further experiments found AIL treatment led to DNA fragmentation, changes in nucleic acid content, cell morphology changes, and decreased cell viability, indicating AIL induces apoptosis in HCT-15 cells. The results suggest AIL has anti-cancer properties and works through an apoptotic
The document describes the design, synthesis, and testing of novel lipopeptide conjugates as potential antimicrobial agents. A library of conjugates was designed based on a template consisting of a hydrophobic moiety, dipeptide, and spermidine. Conjugates containing linoleic acid exhibited potent antibacterial activity against both Gram-positive and Gram-negative bacteria. Structure-activity relationships revealed that optimal activity required a hydrophobicity of 50-70% and a minimum charge of +2. Active conjugates were found to have different modes of action, damaging bacterial cell membranes and DNA. Two conjugates showed selective membrane disruption of pathogenic MRSA and were identified as promising leads for further optimization and development as antimicrobial agents.
Liposomal drug delivery involves encapsulating drugs within liposomes, which are spherical vesicles composed of phospholipid bilayers, to improve drug targeting and reduce toxicity. Liposomes can be classified based on lamellarity, size, and method of preparation. Drugs are encapsulated within the aqueous interior or phospholipid bilayer of liposomes. Liposomes protect drugs, control drug release, and can be targeted to specific tissues. Applications include cancer therapy, antimicrobial delivery, ophthalmic delivery, and topical delivery to improve treatment.
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online
Crimson Publishers-Stable Labeled Isotopes as Internal Standards: A Critical ...CrimsonPublishersMAPP
SIL internal standards are commonly used in LC-MS/MS analyses to improve accuracy and precision. There are two main types: structurally similar analogs or isotopically labeled compounds containing stable isotopes like deuterium, carbon-13, or nitrogen-15. SIL standards provide structural information to understand analyte fragmentation patterns and metabolism. Their use has been shown to reduce ionization variations compared to analog standards. However, SIL standards can still cause ion suppression or enhancement and may not fully correct for matrix effects. While very useful, SIL standards also have limitations like expense and potential for different retention times versus analytes. Overall they remain a preferred choice but alternative standards may still be needed in some cases.
1) The study examined the effects of various cytokinins, cytokinin ribosides, and their analogs on the viability of normal and cancerous human cells.
2) Only a few compounds, including isopentenyladenosine (1a) and N6-benzyladenosine (3a), significantly impaired viability in most cell lines tested, including normal endothelial cells and various cancer cell lines.
3) Colon carcinoma LoVo cells were uniquely insensitive to all compounds tested and may serve as a model for studying cytokinin resistance.
Recent Advancement and Patents of the Lipid Polymer Hybrid Nanoparticlespeertechzpublication
In recent years, robustness and surface engineering of dosage form made improvement in
pharmacokinetics with decrease in dose of drug. Specifi city with adherence of ligands has now become
the reality as surface modifi cation can easily deceive phagocytic system. Lipid molecules ensures the
release of drug at lymphatic system, entrapment of polymeric nanoparticles in lipoidal core led to the
avoidance of disadvantage of low entrapment effi ciency if use of hydrophobic drug with hydrophobic
polymer becomes essential. Various studies have been published and the best formulations with optimal
In vitro and In vivo results are highlighted in this paper. In this review most advanced researches and
accepted patents were discussed so to act as a medium for getting everything regarding lipid polymer
hybrid particles under one umbrella.
This document provides an introduction to biostatistics. It discusses key concepts including descriptive statistics, inferential statistics, hypothesis testing, and sampling techniques. It outlines the role of biostatistics in various areas like clinical medicine, preventive medicine, health planning and evaluation, and medical research. Biostatistics helps manage uncertainties in medicine by providing statistical methods to analyze data, evaluate treatments and programs, and make inferences about populations. It is important for designing valid research studies and interpreting medical literature.
X-rays were discovered in 1895 by Wilhelm Röntgen. X-rays are produced when high velocity electrons hit a metal target, knocking electrons out of inner shells of atoms within the target. As outer electrons fall into these vacancies, characteristic X-rays are emitted with energies specific to the element. X-ray diffraction methods like Bragg's law are used to analyze crystal structures of materials by detecting the angles and intensities of diffracted X-ray beams. Common applications include determining structures of polymers, particle sizes, and states of materials.
This document provides an overview of systematic reviews and meta-analyses. It defines systematic reviews as reviews that use explicit and reproducible methods to minimize bias in identifying, selecting, and synthesizing studies. Key elements of systematic reviews include formulating a clear question, conducting a comprehensive search, applying objective selection criteria, critically appraising included studies, and synthesizing data using meta-analysis if appropriate. Heterogeneity and reporting biases are important considerations in analyzing and interpreting systematic review results. The document outlines the process for conducting high-quality systematic reviews and meta-analyses.
This document provides guidance on critically evaluating biomedical literature. It discusses key elements to examine such as the title, abstract, introduction, objectives, methods, study design, potential biases, statistical analysis, results, discussion and conclusion. The document emphasizes reviewing these sections systematically to determine the validity, relevance and applicability of a study's findings. Selecting high quality literature that could impact clinical practice is important. A thorough evaluation of all aspects of a scientific paper is required to make well-informed judgments.
This document summarizes different types of literature reviews that are commonly used in health research. It discusses 8 major types of reviews: literature review, critical review, scoping review, systematic review, meta-analysis, qualitative systematic review, realist review, and review of reviews. For each type of review, it provides a brief description, discusses their methodology using the Search, Appraisal, Synthesis, Analysis (SALSA) framework, and notes their strengths and weaknesses. The document aims to provide beginners with an overview of different review types and their implications for practice.
This document describes a method for quantitatively separating and estimating lipids in nervous tissue using thin-layer chromatography. The method involves extracting lipids from brain tissue using chloroform and methanol, applying the extract to a thin-layer chromatography plate, separating the lipids using a mobile phase, visualizing the lipids by spraying with sulfuric acid and scanning, and then quantifying the lipids by measuring the area under peaks on a densitometer trace. The method was found to accurately separate and quantify major brain lipids like sphingomyelin, cerebrosides, and phospholipids with standard errors of 2% or less for major lipids.
This document describes the development and validation of a new LC/MS/MS method for quantifying four gangliosides (GM2, GM3, GD2, and GD3) in human plasma. Gangliosides are glycosphingolipids that play important roles in neurological function and disease. The method uses protein precipitation for sample extraction, UPLC separation, and MS/MS detection in MRM mode for high sensitivity and specificity. Product ion mass spectra were obtained for the gangliosides to select optimal MRM transitions for quantification. The method was validated for extraction recovery, calibration linearity, precision, and accuracy. This sensitive assay can be used to monitor ganglioside levels in plasma from normal and
This document discusses the stepwise synthesis of glycolipids in the Golgi complex. It summarizes that glycolipid expression is highly regulated during development through transcriptional control of key glycosyltransferases. These transferases reside in the Golgi and depend on N-glycosylation and oligosaccharide processing for proper folding. Their N-terminal domain transports them to the Golgi where some associate to form multienzyme complexes. The precise organization and dynamics of this machinery in the Golgi is a potential target for fine-tuning glycolipid expression.
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Hydrophilic interaction liquid chromatography (HILIC) is a powerful technique for separating polar compounds. The document reviews options for characterizing HILIC stationary phases and their applications. HILIC employs polar stationary phases like silica, amino, or cyano bonded phases. Retention depends on factors like the stationary phase properties, mobile phase composition, and analyte structure. HILIC has advantages over normal and reversed phase chromatography and is useful for separating compounds that are too polar for reversed phase methods.
This document provides an overview of the role of process validation in tablet manufacturing. It discusses that process validation is required to demonstrate that a manufacturing process is capable of consistently producing tablets that meet predetermined specifications and quality attributes. The key aspects of process validation covered include the types of validation, phases of validation, critical factors, and use of sample thieves. Process validation helps assure quality, reduce costs from rejects and failures, and ensures compliance with regulatory requirements. It is an important part of good manufacturing practices for pharmaceutical products.
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TEST BANK For Accounting Information Systems, 3rd Edition by Vernon Richardson, Verified Chapters 1 - 18, Complete Newest Version
TEST BANK For Accounting Information Systems, 3rd Edition by Vernon Richardson, Verified Chapters 1 - 18, Complete Newest Version
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2. ment of software tools for parameter optimization, identi-
fication, quantification, and visualization, such as Multiquant15
and Skyline-modified “lipidomics”,16
has made it possible to
analyze many different sphingolipid species. However, many
isobaric and nearly isobaric sphingolipids cannot be differ-
entiated from one another using a low-resolution/low-mass
accuracy triple-quadrupole mass spectrometer and/or relying
on single transitions as the identifier and quantifier, even with
sophisticated LC separations. To obtain a more precise picture
of which molecules are present in a sample, the analysis of
sphingolipids should be conducted by monitoring the exact
mass of the precursor and multiple fragments, including the
long-chain base (LCB), fatty acyls, and/or class specific
fragments, thereby allowing annotation at the fatty acyl species
level.17
Therefore, we developed an improved workflow that
includes an optimized extraction procedure, a segmental linear
chromatographic gradient, and a high-resolution (HR)/high-
mass accuracy full scan/parallel reaction monitoring (PRM)
approach for a comprehensive sphingolipid analysis. This
enabled us to profile sphingolipids in response to autophagy in
RAW 264.7 cells. The lipid profile of this cell line is published,
and the cells are used by the LIPID MAPS consortium as a
standard cell line for method development, enabling us to apply
a standardized cell culture protocol and directly benchmark our
method.18
■ EXPERIMENTAL SECTION
Materials. Chemicals and reagents were obtained from the
following sources: formic acid, ammonium formate, acetic acid
(HAc), potassium hydroxide (KOH), MS-grade phosphoric
acid (85−90%), and tert-butyl methyl ether (MTBE) from
Sigma-Aldrich (Steinheim, Germany); MS-grade acetonitrile
(ACN) and methanol (MeOH) from Biosolve (Valkenswaard,
The Netherlands); sodium chloride (NaCl) and isopropanol
(IPA) from Merck (Darmstadt, Germany); sodium dodecyl
sulfate (SDS) from Roth (Karlsruhe, Germany); tris-
(hydroxymethyl)aminomethane (Tris) from Applichem
(Darmstadt, Germany); and Kdo2-Lipid A (KLA), monosulfo
galactosyl ceramide (GalaCerS) d18:1/12:0, and ceramide/
sphingoid internal standard mixture II (CerMix) consisting of
sphingosine d17:1, sphinganine d17:0, sphingosine-1-P d17:1,
sphinganine-1-P d17:0, sphingomyelin d18:1/12:0, ceramide
(Cer) d18:1/12:0, glucosylceramide d18:1/12:0 (GlcCer,
internal standard for HexCer), lactosylceramide (LacCer, as
the internal standard for DiHexCer) d18:1/12:0, and ceramide-
1-P (CerP) d18:1/12:0 in ethanol from Avanti Polar Lipids
(Alabaster, AL). Ultrapure water (18 MΩ cm at 25 °C) was
obtained from an Elga Labwater system (Lane End, U.K.). The
bicinchoninic acid assay (BCA) was obtained from Thermo
Fisher Scientific (Rockford, IL).
Cell Culture. Mouse macrophages (RAW 264.7; ATCC,
Manassas, VA) were treated with KLA according to the
protocol provided by LIPID MAPS (www.lipidmaps.org/
protocols); the detailed protocol is described in the Supporting
Information. Three biological replicates each for untreated and
treated cells were prepared from a single batch of cells. The cell
pellets were transferred into a 2 mL polypropylene tube
(Eppendorf, Hamburg, Germany) prior to extraction.
Western Blot. Western blot was performed as described by
Barth et al.36
Further information can be found in the
Supporting Information.
Sample Preparation. Four different extraction methods
without and with alkaline treatment were tested to identify the
extraction protocol enabling the best determination of low-
abundance sphingolipid species. Alkaline hydrolysis of the
esters was applied to reduce the chromatographic interference
of glycerophospholipids and glycerolipids.19
Chloroform/
methanol (CHCl3/MeOH) extraction and alkaline CHCl3/
MeOH extraction were conducted as previously described by
Shaner et al.12
Detailed information is given in the Supporting
Information. MTBE extraction was performed following the
protocol of Matyash et al.20
(Supporting Information). Alkaline
MTBE extraction is based on the MTBE protocol of Matyash
et al.20
with small modifications. After the first incubation, 97.5
μL of 1 M KOH in MeOH was added and the mixture
incubated in the thermomixer for 2 h at 37 °C. After the
mixture was cooled to 25 °C, 2 μL of HAc was added to
neutralize the solution. Afterward, 188 μL of water was added,
and the samples were centrifuged at 10000g for 10 min at 4 °C.
The lipid extract was stored at −80 °C prior to further analysis.
High-Performance Liquid Chromatography (HPLC)
Gradients. Gradients previously utilized for sphingolipid
analysis were chosen for comparison21−23
(Table S1). In
addition, a segmented linear gradient was designed with
GradientOptimizer by inputting the retention times of
sphingolipid species that were obtained from a linear gradient
(initial 30% B, held at 30% B from 0.0 to 3.0 min, 30 to 75% B
from 3.0 to 15.0 min, 75 to 100% B from 15.0 to 17.0 min, and
100% B from 17.0 to 25.0 min).24
LC−MS/MS. Evaluation of Extraction Protocols. Initial
extraction comparison was accomplished with a QTRAP 6500
instrument (Applied Biosystems, Darmstadt, Germany) that
was equipped with an electrospray ion source (Turbo V ion
source). The detailed information about ESI source settings is
provided in the Supporting Information. The collision energy
was optimized for each sphingolipid class by direct infusion of a
corresponding standard. The scheduled SRM detection window
was set to 2 min, and the cycle time was set to 2 s. Data were
acquired with Analyst version 1.6.2 (AB Sciex, Concord, ON).
Skyline (64-bit, 3.5.0.9319) was used to calculate the lipid
transition list, visualize results, integrate signals over the time,
and quantify all lipids that were detected by MS.16
Sphingolipid Analysis. High-performance liquid chromatog-
raphy−electrospray ionization tandem mass spectrometry
(HPLC−ESI-MS/MS) was conducted using an UltiMate
3000 instrument (Thermo Fischer Scientific, Darmstadt,
Germany) in conjunction with a Q Exactive Plus mass
spectrometer (Thermo Fisher Scientific, Bremen, Germany).
Chromatographic separation was accomplished with an
Ascentis Express C18 column (150 mm × 2.1 mm, 2.7 μm;
Supelco, Bellefonte, PA) fitted with a guard cartridge (50 mm ×
2.1 mm, 2.7 μm; Supelco). The temperatures of the
autosampler and the column oven were set at 10 and 60 °C,
respectively. All gradients and LC parameters can be found in
Table S1. The injector needle was automatically washed with
30% B and 0.1% phosphoric acid prior to each injection.
Samples were injected in a volume of 5 μL. The use of
phosphoric acid did not affect LC or MS instrumentation over a
running period of three years. The Q Exactive Plus instrument
was configured to perform a high-resolution MS full scan (HR-
FS) and PRM in one measuring cycle. Full scan settings were as
follows: m/z 250−900 [positive ion detection, resolution of
70000 (m/z 200)]; AGC (automatic gain control), 3 × 106
;
maximum injection time, 50 ms. For PRM, spectra were
acquired as follows: isolation windows, m/z 0.5 [mass
resolution of 17500 (m/z 200); AGC, 1 × 105
; maximum
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B
3. injection time, 100 ms]. The normalized collision energy
(NCE) was optimized for each sphingolipid class by direct
infusion of a corresponding standard. An inclusion list of
precursors was used for scheduled PRM data acquisition
(Tables S2 and S3).
■ RESULTS AND DISCUSSION
SRM of lipid extracts using a triple-quadrupole mass
spectrometer has been the method of choice for targeted
sphingolipid analysis. However, technical limitations of the
instrumentation in combination with the high level of
complexity of lipid extracts compromise accurate identification
and quantification of sphingolipids. The major issues associated
with this strategy are interference (ion suppression and isotope
overlapping) from abundant phospholipids, nearly isobaric
sphingolipid species that cannot be distinguished on the basis
of low resolution masses, common fragment ions derived from
key structural features (such as the LCB), the overlap of natural
isotope clusters due to varied degrees of unsaturation among
isobars, and chromatographic co-elution of isobaric sphingoli-
pids. To overcome these challenges, we present here an
improved workflow that includes an optimized extraction
procedure, a segmental linear HPLC gradient, and a HR-FS/
PRM approach, resulting in comprehensive sphingolipid
analysis (Figure 1).
Establishment of a Comprehensive Profiling Strategy
for Sphingolipids. Comparison of Extraction Methods for
Sphingolipids. An effective extraction strategy is essential for
obtaining a comprehensive sphingolipid profile. Over five
decades, CHCl3/MeOH extraction has been the most widely
used protocol for lipid analysis,25
although several modified
methods have been reported for selected sample types and lipid
classes.26−28
In recent years, MTBE extraction has been shown
to deliver similar or even better recoveries of most lipid species,
including sphingolipids.20
However, original protocols were
designed to isolate all lipid classes together, with the
consequence that there are frequently false positive identi-
fications for sphingomyelin (SM) due to the presence of
glycerophosphatidylcholines that have the same phosphocho-
line headgroup and overlap with the natural isotope pattern of
SM when analyzed on a triple-quadrupole mass spectrometer.
An alkaline hydrolysis step has been used in conjunction with
CHCl3/MeOH or MTBE extraction as a way to hydrolyze all
ester bonds and deplete the samples of phospholipids and/or
glycerolipids as well as reduce matrix effects.12,20
Because of an
increased solubility in water, the hydrolyzed fragments are not
retained on the stationary phase. To evaluate the consequences
of alkaline hydrolysis, we benchmarked four different extraction
methods against each other for extraction of sphingolipids from
RAW 264.7 cells: CHCl3/MeOH extraction with and without
alkaline hydrolysis and MTBE with and without alkaline
hydrolysis. We chose SM d18:1/16:0, a common sphingomye-
lin species, to demonstrate the resolution of overlap with PC
species. Two characteristic fragments were selected for
monitoring: the phosphocholine headgroup (m/z 184.1) and
the LCB (m/z 264.3). While there was no significant difference
in the intensity of the LCB fragment because of the inclusion of
alkaline hydrolysis, the chromatographic traces of the
phosphocholine fragment derived from SM were considerably
improved by eliminating co-eluting PCs (Figure S1A,B). As can
be seen from the total ion chromatogram (TIC) derived from
the high-resolution full scan (HR-FS) in Figure 2, hundreds of
co-eluting phospholipids and glycerolipids between 6 and 10
min were eliminated by alkaline hydrolysis.18
Although the
Figure 1. Integrated approach for sphingolipid profiling, including (A) alkaline extraction for sphingolipids, (B) an optimized gradient for LC
separation, (C) parallel reaction monitoring and full scan for MS scanning, and (D) critical criteria for identification.
Figure 2. Comparison of total ion chromatograms (TICs) of (A) non-alkaline and (B) alkaline MTBE extraction for sphingolipid analysis.
Abbreviations: PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PG, phosphatidylglycerol;
DG, diacylglycerol; TG, triacylglycerol; SM, sphingomyelin; Cer, ceramide; HexCer, hexosyl ceramide.
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C
4. alkaline solution hydrolyzes amides at a slower rate, precautions
should be taken and experiments carefully planned and
designed. On the basis of a comparison of internal standards
with and without base hydrolysis, no significant hydrolysis was
observed for the main sphingolipid classes Cer, HexCer,
DiHexCer, and SM (Figure S2). A notable advantage of the
MTBE protocol is that it is less time-consuming than CHCl3/
MeOH extraction (only ∼4 h for the preparation of lipid
extracts compared to at least 12 h), and it permits easier
collection of the organic phase and precipitated proteins.29
Quantitative results obtained from inclusion of internal
standards indicated that MTBE alone or combined with the
alkaline treatment consistently resulted in an extraction
efficiency that is better than that of CHCl3/MeOH or
CHCl3/MeOH extraction with alkaline treatment for the
more hydrophobic species with longer fatty acids such as SM
d18:1/24:0 (7859 ± 614.7 pmol/mg of protein vs 886.9 ±
88.18 pmol/mg of protein) and Cer d18:1/24:0 (1890 ± 169.9
pmol/mg of protein vs 189.7 ± 29.91 pmol/mg of protein).
For relatively hydrophilic species such as So 18:1, MTBE
extraction showed the same extraction ability as CHCl3/MeOH
extraction (Figure S1C). The results of CHCl3/MeOH
extraction with alkaline hydrolysis yielded the same concen-
tration as MTBE with base hydrolysis (Figure S1C), and
MTBE extraction with or without base hydrolysis exhibited an
overall lower level of variability. Addition of a base hydrolysis
step to the MTBE method eliminates lipid species that co-elute
with sphingolipids by RP-HPLC, thereby reducing the level of
background interference and yielding better signal-to-noise
ratios (Figure S1A,B), which is particularly important for
identification and quantification of low-abundance sphingoli-
pids. As such, this is the extraction method of choice for
sphingolipid analysis. The preliminary sphingolipid analysis was
accomplished by scanning known sphingolipid species in RAW
264.7 cells in SRM mode using a QTRAP 6500 instrument.
Although extraction efficiency is independent of the mass
spectrometer used for the analysis, SRM-based quantification
on a triple quadrupole is limited for nearly isobaric species. It is
also challenging to scan for all potential lipid-derived fragment
ions of sphingolipids due to a limitation in the number of
transitions that can be monitored in SRM mode. Furthermore,
the lack of high-resolution/high-mass accuracy information for
the precursors analyzed on a triple-quadrupole instrument can
hinder correct interpretation of results. Therefore, subsequent
investigations were accomplished with HRMS by applying FS
and PRM.
Comparison of HPLC Methods for Sphingolipid Analysis.
An efficient HPLC separation is essential for increasing the
specificity and sensitivity of detection of sphingolipids. On the
basis of the results of previous studies,21−23
we evaluated
different reversed-phase separation strategies that are currently
used in the field for sphingolipid analysis (Figure 3). The
results from two (Table S1) of these are reported here. All
three combinations performed similarly for HexCer and
Figure 3. Comparison of LC methods for sphingolipid separation. Panels from left to right present ion chromatograms at the precursor and fragment
level. Mass errors are indicated with color-coded text next to corresponding signals. For analysis, reversed-phase chromatography [Ascentis Express
C18 column (150 mm × 2.1 mm, 2.7 μm)] and full scan PRM methods on an Q Exactive Plus instrument are applied. (A) Gradient I used MeOH/
IPA/H2O solvent compositions from ref 23 to separate sphingolipids. (B and C) Gradients II and III used the same ACN/IPA/H2O solvent
composition but different gradients. The parameters and the gradient composition are listed in Table S1. ① indicates lipid species Cer d18:1/24:1,
while ② indicates Cer d18:2/24:0.
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D
5. DiHexCer species, while I [resolution (R), 100%] and III (R,
115−409%) yielded separation that was better than that of II
(R, 91−230%) for ceramides. III offered baseline separation
that was better than that of I for Cer d18:1/24:1 and Cer
d18:2/24:0 (R, 116%). III also offered the best separation for
SM with the least co-elution of isobaric species, as verified by
the elution pattern of characteristic SM fragments and R
(Figure 4 and Figures S3 and S4). The results of our HPLC
gradient evaluations indicated that use of a segmented linear
gradient (III) is optimal and faster for sphingolipid separation
and is likely to be an effective option for lipidomics in general.
MS Approach for Sphingolipid Profiling. The use of a high-
resolution/high-mass accuracy mass spectrometer is particularly
important for assuring the validity of targeted sphingolipid
analyses because of the presence of multiple nearly isobaric
species (Figure 5A). For example, differentiation of Cer d18:1/
23:0 [molecular weight (MW) of 635.6216] from Cer d18:1/
h22:1 (MW of 635.5852) requires a resolving power of >17000
and a mass accuracy better than 57 ppm. However, even though
the two nearly isobars can be clearly identified in MS1, they
fragment together because of the 0.5 Da isolation window
during precursor ion selection in PRM. Because there are
multiple product ions in the majority of MS2 spectra of
sphingolipids (Table S4 and Figure S5), the use of
fragmentation patterns in conjunction with an accurate mass
is the most effective way to accurately identify a sphingolipid
species (Figure 5B). SRM has been the method of choice in
sphingolipid analyses, but use of SRM can be problematic
because the full MS2 profile is not acquired. In particular, if
only the transition generating the LCB is monitored on a triple-
quadrupole mass spectrometer, it is not possible to detect the
presence of nearly isobaric sphingolipids (e.g., HexCer d18:1/
16:0 and CerP d18:1/22:1 that contain the same LCB
fragment). This can be remedied through the use of PRM.
The complete fragmentation pattern (Figures S5 and S6) can
be utilized for confident identification within an individual
sphingolipid class, supporting the distinction of isobaric
precursors such as Cer d18:1/22:0 and Cer d18:0/22:1 (Figure
5C). The existence of fragment-level isobars with overlapping
isotopes is an additional confounding problem that cannot be
solved chromatographically. As such, a targeted method was
established in concert with high-resolution MS to detect and
quantify the various molecular species (Figure 5D).
Confident identification of sphingolipids at fatty acid scan
species level17
requires the use of at least two or three
fragments: the class specific fragment (if available), the LCB,
and the fatty acid to unambiguously identify the lipid molecule.
High-resolution survey scans in conjunction with PRM offer
the possibility of determining the exact mass of the intact
sphingolipid and a complete MS2 spectrum to validate the
sphingolipid identity. For identification, it is essential that the
mass errors of precursor ions and/or fragment ions be below 5
ppm and that there be a match of the fragmentation pattern to
that of an authentic standard. For Cer, HexCer, DiHexCer,
sulfatides, and gangliosides, a group of fragments is associated
with the LCB (Figure S5, red font). We call this group the
“LCB pattern”, with the fragments designated as W′, W″, and
W′-CHO. These fragments are generated in the course of HCD
(higher-energy collisional dissociation) and can be used to
identify sphingolipids using MS2 spectra (Figures S4 and S8).
Knowledge that the ratios of fragment intensities (W′:W″:W′-
CHO) should be approximately 10:100:15 for an unsaturated
LCB and 2:50:100 for a saturated LCB is important for
determining if there is co-isolation or co-elution of any
sphingolipid species (Figure S5 and Table S5). The ratio
between different transition intensities, the so-called qualifier
ion ratio (QIR), is used to support unequivocal molecule
verification.30,31
The known QIR value (derived from analysis
of an authentic standard) should always be achieved by well-
separated lipids. It is worth noting, on the basis of analysis of
authentic standards, we have found that this set of fragments is
not generated for SM and CerP. Therefore, for SM and CerP,
along with the exact mass and the LCB fragments, additional
ions are required to identify and differentiate these two lipid
classes from other sphingolipids: the phosphocholine head-
group for SM (m/z 184.0733) and neutral loss of the
phosphate group of CerP (loss of 97.9769 Da) (Figure S7).
For SM identification, a further fragment can be observed when
the correct collision energy is used (34 V for HCD on our Q
Exactive Plus instrument). Under these conditions, a low-
intensity fatty acid carboxylate anion [m/z 199.1705 for SM
d18:1/12:0 (m/z 199.1698 calculated, +3.5 ppm) and m/z
255.2330 for SM d18:1/16:0 (m/z 255.2324 calculated, +2.4
ppm)] is detected in negative ion mode (Figure S8). This
fragment is presumably generated via an intramolecular
rearrangement (retro-heteroene reaction) involving the oxygen
on the LCB, because the close agreement between calculated
and observed masses clearly indicates the presence of two
oxygens.32
Although the intensity for the carboxylate fragment
is relatively low, it is detectable for medium- to high-abundance
species (such as SM d18:1/16:0) and provides valuable
evidence for structure verification.
Sphingolipid Profile in RAW 264.7 Cells. To illustrate
the effectiveness of our sphingolipid workflow, we analyzed the
sphingolipids in RAW 264.7 cells and assessed the effect of
KLA on sphingolipid metabolism. KLA treatment of RAW
264.7 cells has been previously reported to induce autophagy
and to increase the levels of Cer by specifically activating the
Toll-like 4 receptor (TLR-4).33,34
During autophagy, micro-
tubule-associated protein 1 light chain 3 (LC3) is involved in
the formation of autophagosomal membranes. LC3 has two
Figure 4. Comparison of LC methods for sphingomyelin. (A) Ion
chromatogram of the d18:1 LCB for SM d18:1/16:0 (①) and
interfering precursor ion (②). Mass errors are colored red. (B) PRM
MS/MS spectrum of ① and ②.
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6. Figure 5. Current challenges in sphingolipid analysis. (A) In challenge I, nearly isobaric species from low-resolution- and mass accuracy-derived data,
e.g., from triple-quadrupole MS, are not always sufficient to resolve closely related species. (B) In challenge II, near isobars are sharing fragment ions.
The chemical formulas (C40H83ON2P3, C43H76O5N2, C44H76O6, and C43H77O3N2P) are examples that also fit the detected precursor masses within a
5 ppm mass error. Collecting multiple fragments rather to rely on a single diagnostic fragment reduces false positive identification. Liquid
chromatographic separation is required. (C) In challenge III, isobars overlap with isotopes. The PRM experiment via high-resolution MS showed
disagreement with SRM via triple-quadrupole MS. The PRM overcomes SRM limitation by providing full MS2 spectra with multiple fragments with
high resolution and mass accuracy to calculate transition intensity ratios (QIRs) by peak areas (a and b). (D) In challenge IV, isobaric species are at
the precursor and fragment level. This challenge can be overcome only with the best possible resolution at LC, a full scan, and a PRM approach.
Abbreviations: M, exact mass; EIC, extracted ion chromatogram.
Figure 6. KLA-induced autophagy in RAW 264.7 cells. (A) The unmodified and lipidated version of LC3 (LC3-II), as autophagy protein markers, is
detected by Western blot. Tubulin staining serves as a loading control for the LC3-II protein. (B) Fold change in the relative amount of LC3-II in
control and KLA-treated cells (based on densitometric quantification of three biological replicates). (C) Selected regulated sphingolipid species. All
sphingolipids are listed in Figure S9. All experiments were performed in biological triplicate. *p < 0.05.
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Anal. Chem. XXXX, XXX, XXX−XXX
F
7. forms, cytosolic LC3-I and membrane-bound LC3-II.35
LC3-II
is a lipidated form of LC3 that has been shown to be an
autophagosomal marker in mammals, providing evidence that
autophagy is induced after KLA treatment.33
We monitored
lipidated LC3-I and LC3-II by Western blot after KLA
treatment and used tubulin as a loading control.36
Our results
are in good agreement with earlier studies that observed an
increase in the level of LC3-II (Figure 6B) and an increase in
the LC3-II:LC3-I ratio between treatment and control (Figure
6A). This indicates induction of autophagosome formation in
KLA-treated RAW264.7 cells. We found that the levels of most
of the Cer species were increased after KLA treatment relative
to SM (Figure 6C and Figure S9). Through use of our
workflow, we identified more sphingolipid species with high-
quality spectra and high confidence than in previous
reports.12,18
We were able to detect and quantify 50% more
Cer species than earlier studies did.12,18
Cer levels were also
altered after KLA treatment (Figures 6C and 7). The number
of HexCer, SM, and CerP species identified by our workflow is
technically lower than in other reports. However, there is a high
level of confidence associated with our identifications, because
they are based on high-resolution/high-mass accuracy precursor
data and the complete tandem mass spectral pattern. In
contrast, only the LCB (i.e., one fragment) that is shared
among many sphingolipid species was used for sphingolipid
identification in the referenced papers. Use of the sphingolipid
fragmentation pattern in combination with high-resolution/
high-mass accuracy precursor measurements increases the
reliability substantially, because many false positive lipids are
removed from consideration. In total, we identified and
quantified 61 sphingolipid species within a dynamic range of
7 orders of magnitude with detection ranging to the low
femtomole per milligram protein level. In agreement with
earlier studies of the same cell type,18,37
sulfatides were not
identified after KLA treatment.
■ CONCLUSIONS
A RPLC/HRMS-based workflow has been developed that
significantly increases the accuracy in sphingolipidomics by
resolving isobaric and nearly isobaric species through use of an
efficient MTBE-based extraction approach that includes alkaline
hydrolysis that removes interfering lipids, a segmented linear
gradient that has been tailored for sphingolipids and effectively
separates closely eluting sphingolipid species, and an MS
strategy that includes HRMS survey scans and PRM MS
detection. Our workflow provides enhancements in sample
preparation and analysis that yield a higher level of confidence
for sphingolipid identification and quantification. Application of
the workflow to analysis of sphingolipids in RAW 264.7 cells
demonstrates its utility for samples of biological origin.
■ ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.anal-
chem.7b03576.
Tables S1−S5, Figures S1−S9, and additional informa-
tion as mentioned in the text (PDF)
■ AUTHOR INFORMATION
Corresponding Author
*Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V.,
Otto-Hahn-Str .6b, 44227 Dortmund, Germany. Phone: +49-
231-1392-4173. Fax: +49-231-1392-4173. E-mail: robert.
ahrends@isas.de.
ORCID
Cristina Coman: 0000-0002-3771-2410
Dominic Winter: 0000-0001-6788-6641
Robert Ahrends: 0000-0003-0232-3375
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This study was supported by the Ministerium für Innovation,
Wissenschaft und Forschung des Landes Nordrhein-Westfalen,
the Senatsverwaltung für Wirtschaft, Technologie und For-
schung des Landes Berlin, and the Bundesministerium für
Bildung und Forschung and by the BMBF de.NBI program
(code 031L0108A). The authors thank Dr. Christian Hellmuth
for valuable comments on the manuscript.
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