Porcine Brain Extracts Research Articles

FAIMS Shotgun Lipidomics for Enhanced Class- and Charge-State Separation Complemented by Automated Ganglioside Annotation.

The analysis of gangliosides is extremely challenging, given their structural complexity, lack of reference standards, databases, and software solutions. Here, we introduce a fast 6 min high field asymmetric ion mobility spectrometry (FAIMS) shotgun lipidomics workflow, along with a dedicated software solution for ganglioside detection. By ramping FAIMS compensation voltages, ideal ranges for different ganglioside classes were obtained. FAIMS revealed both class- and charge-state separation behavior based on the glycan headgroup moiety. The number of sialic acids attached to the glycan moiety correlates positively with their preferred charge states, i.e., trisialylated gangliosides were mainly present as [M - 3H]3- ions, whereas [M - 4H]4- and [M - 5H]5- ions were observed for GQ1 and GP1. For data evaluation, we developed a shotgun/FAIMS extension for the open-source Lipid Data Analyzer (LDA), enabling automated annotation of gangliosides up to the molecular lipid species level. This extension utilized combined orthogonal fragmentation spectra from CID, HCD, and 213 nm UVPD ion activation methods and covers 29 ganglioside classes, including acetylated and fucosylated modifications. With our new workflow and software extension 117 unique gangliosides species were identified in porcine brain extracts. While conventional shotgun lipidomics favored the observation of singly charged ganglioside species, the utilization of FAIMS made multiply charged lipid species accessible, resulting in an increased number of detected species, primarily due to an improved signal-to-noise ratio arising from FAIMS charge state filtering. Therefore, this FAIMS-driven workflow, complemented by new software capabilities, offers a promising strategy for complex ganglioside and glycosphingolipid characterization in shotgun lipidomics.

Manipulation of Ion Types via Gas-Phase Ion/Ion Chemistry for the Structural Characterization of the Glycan Moiety on Gangliosides.

Gangliosides are the most abundant glycolipid among eukaryotic cell membranes and consist of a glycan head moiety containing one or more sialic acids and a ceramide chain. The analysis of the glycan moieties among different subclass gangliosides, including GM, GD, and GT gangliosides, remains a challenge for shotgun lipidomics. Here, we present a novel shotgun lipidomics approach employing gas-phase ion/ion chemistry. The gas-phase derivatization strategy provides a rapid way to manipulate the ion-types of the precursor ions, and, in conjunction with collision induced dissociation (CID), allows for the elucidation of the structures of the glycan moieties from gangliosides. In addition to the enhancement of structural characterization, gas-phase ion chemistry leads to a form of purification of the precursor ions prior to CID by neutralizing isobaric or isomeric ions with different charge states but with similar or identical m/z values. To demonstrate the proposed strategy, both deprotonated GM3 and GM1 gangliosides ([GM-H]-) were isolated and subjected to reaction with magnesium-Terpy complex cations ([Mg(Terpy)2]2+). The post-reaction product spectra show the elimination of possible contamination, illustrating the ability of charge-switching derivatization to purify the precursor ions. Isomeric differentiation between GD1a and GD1b was achieved by the sequential ion/ion reactions, with the CID of [GD1-H+Mg]+ showing diagnostic fragment ions from the isomers. Moreover, isomeric identification among GT1a, GT1b, and GT1c was accomplished while performing a gas-phase magnesium transfer reaction and CID. Lastly, the presented workflow was applied to ganglioside profiling in a porcine brain extract. In total, 34 gangliosides were profiled among only 20 precursor ion m/z values by resolving isomers. Furthermore, the fucosylation site on GM1 and GD1, and N-glycolylneuraminic acid conjugated GT1 isomers was identified. Relative quantification of isomeric two isomeric pairs, GD1a/b C36:1 and GD1a/b C38:1 was also achieved using pure component product ion spectra coupled with a total least-squares method. The results demonstrate the applicability and strength of using shotgun MS coupled with gas-phase ion/ion chemistry to characterize the glycan moiety structures on different subclasses of gangliosides.

Lipidomic analysis using hydrophilic interaction liquid chromatography microgradient fractionation of total lipid extracts

Lipidomic samples are complex mixtures of structurally different species of a wide range of concentrations providing challenges in their characterization. In this work, we present a proof of concept for the application of a simple microgradient liquid chromatography device on the detailed analysis of lipid classes. Our lipidomic analysis is based on a lipid class microgradient fractionation of a total lipid extract using an in-house-prepared hydrophilic interaction liquid chromatography microcolumn followed by RP-LC/MS of the collected lipid class fractions. The final fractionation method uses a 40-mm-long microcolumn of 500 μm ID with silica stationary phase obtained from a commercially available chromatographic column and the microgradient of the mobile phase prepared in a microsyringe using methyl tert-butyl ether (MTBE) – methanol – water – ammonium acetate mixtures of various elution strengths. MTBE total lipid extract is directly separated by microgradient elution into lipid classes according to their polarity, which enables the collection of isolated fractions of most lipid classes. The method has been applied to the fractionation of porcine brain extract into nonpolar lipids, hexosylceramides, phosphoethanolamines, phosphocholines, sphingomyelins, and lysophosphocholines classes. Achieved repeatability, recovery, and advanced lipid coverage prove the applicability of the microgradient fractionation of total lipid extract for the comprehensive lipidomic analysis.

Detailed Characterization of the Postionization Efficiencies in MALDI-2 as a Function of Relevant Input Parameters.

A recently introduced technique based on MALDI with laser-induced postionization (PI), also named MALDI-2, increases the ion yields for numerous classes of lipids, metabolites, and carbohydrates in MALDI-MS imaging experiments under certain experimental conditions. Here, we used a semiautomatic LabVIEW-based protocol to investigate and optimize the efficiency of the PI process dependent on four relevant input parameters and a dense parameter grid: pulse energies of the two lasers, delay between the laser pulses, and buffer gas pressure in the ion source. All experiments were conducted with a modified MALDI-2 Synapt G2-S mass spectrometer (Waters) and use of a focal spot size on the sample of 15-17 μm. A wavelength-tunable optical parametric oscillator (OPO) laser served for PI at 260 or 280 nm. The investigated MALDI matrices were: 2,5-dihydroxybenzoic acid (positive ion mode, +), 2,5-dihydroxyacetophenone (+), α-cyano-4-hydroxycinnamic acid (+), norharmane (negative-ion mode, -), and 1,5-diaminonapthalene (-). A porcine brain extract served as lipid standard. In the positive-ion mode, a maximum boost for the generated [M + H]+ species was found with a N2 buffer gas pressure of ∼2 mbar and a delay between the laser emissions of ∼10 μs. Higher optimal delay settings of several 10 μs were registered for the two studied matrices in negative-ion mode. With regard to the laser fluences, best PI efficiencies were reached using maximum available ablation and PI laser pulse energies of up to 25 and 160 μJ, respectively. For analytes not profiting from MALDI-2, best ion signal yields were recorded for ablation laser pulse energies of around 7 μJ, depending on the MALDI matrix. At higher laser pulse energies, sizable fragmentation is observed for these ions. The PI laser pulse energy did not have any influence on the ion signals of these species. For optimal ion yield of all analyte species, best results were obtained with an ablation laser pulse energy of ∼7 μJ and a PI laser pulse energy of ∼160 μJ. Our comprehensive data set provides valuable insight into the mechanisms underlying the MALDI-2 processes and could help to further optimize this emerging technique.

Effects of porcine brain hydrolysate on impairment of cognitive learning ability in amyloid β(1-40) -infused rats.

This study assessed whether administering porcine brain hydrolysate (PBH) ameliorates the impairment of spatial cognition learning ability in amyloid β (Aβ)-infused rats. PBH was prepared using organic solvents (i.e., acetone and ethanol). Enzyme hydrolysates were derived from these PBH and the sequence of the Aβ peptide for infusion was selected. The results indicated the PBH, in particular EP (porcine brain extract with ethanol and protease N), demonstrated the potentials to reduce damage of neurodegenerative disorders invitro and invivo. The principal findings of this study indicate that PBH has prolyl endopeptidase inhibitory activity invitro. Moreover, administering EP to Aβ(1-40)-infused rats significantly improves their performance on reference, spatial performance, and working memory tests during water maze tasks; concurrent proportional decreases are also observed in malondialdehyde levels, acetylcholinesterase (AChE) activity, and Aβ accumulation levels in brain tissues. The PBH was suggested to ameliorate learning deficits associated with Alzheimer's disease by inhibition of lipid peroxidation in the brain of Aβ infused rat.

Porcine brain extract promotes osteogenic differentiation of bone marrow derived mesenchymal stem cells and bone consolidation in a rat distraction osteogenesis model

Distraction osteogenesis (DO) is the gold standard to treat large bone defects, but long consolidation period is a major limitation. Innovative efforts to promote osteogenesis are needed. Porcine brain extract (PBE) was reported to enhance the proliferation and differentiation of multiple primary cells. In this study, we aimed to develop a method for collecting PBE and investigate its effects on osteogenic differentiation of rat bone marrow derived mesenchymal stem cells (rBMSCs) and bone consolidation in a rat DO model. The PBE was collected from neonatal brain tissues of porcine fetus and was used to treat rBMSCs. Following PBE treatment (700 ng/ml), osteogenic differentiation was assessed. Further, we locally injected PBE (7 μg/ml, 100μl) or PBS (100μl) into the gap in a Sprague-Dawley (SD) male rat DO model every three days till termination. X-rays, micro-computed tomography, mechanical testing, histology and immunohischemistry examinations were used to exam the quality of the regenerates. The alkaline phosphatase, calcium deposits, and steogenic markers in the PBE treated rBMSCs were significantly increased. In the rat model, new bone properties of bone volume/total tissue volume and mechanical strength were higher in the PBE treated group. Histological analysis also confirmed more mineralized bone after PBE treatment. The current study reports a standard protocol for PBE collection and demonstrated its positive effects on osteogenic differentiation and bone consolidation in DO. Since the PBE is readily available and very cost effective, PBE may be a potential new bio-source to promote bone formation in patients undergo DO treatment.

Enrichment of Metabolite-Binding Proteins by Affinity Elution in Tandem Hydrophobic Interaction Chromatography (AETHIC) Reveals RKIP Regulating ERK Signaling in an ATP-Dependent Manner.

To elucidate the molecular mechanisms underlying the action of bioactive compounds such as metabolites, identification of their binding targets is essential. However, available techniques for enriching metabolite-binding proteins are practically restrained by special equipment requirements and laborious efforts. Here we have developed a novel method, affinity elution in tandem hydrophobic interaction chromatography (AETHIC), which enables enrichment of metabolite-binding proteins from a crude tissue extract. AETHIC constitutes two major steps, protein fractionation and affinity elution. The basic strategy of AETHIC uses a series of HIC matrices encompassing aliphatic chains of different length and thus provides a wide range of hydrophobicity for interactions with most proteins. Thereafter, target proteins are eluted selectively by a given ligand. As our first proof-of-principle, we demonstrated that AETHIC was able to enrich ATP-binding proteins from porcine brain extract. In addition, we have demonstrated that raf kinase inhibitory protein (RKIP) is an ATP-binding protein and ATP attenuates the interaction between RKIP and Raf-1. In parallel, short-term ATP depletion in cultured HEK293 cells augments interaction between RKIP and Raf-1, resulting in decreased activation of the downstream ERK signaling. Therefore, the ATP-binding function renders RKIP's inhibition on Raf-1 modulated by cellular ATP concentrations. These data shed light on how energy levels affect the propagation of cellular signaling. Taken together, the enclosed results advocate the potential of AETHIC in the study of metabolite-protein interactions.

Retention behavior of lipids in reversed-phase ultrahigh-performance liquid chromatography–electrospray ionization mass spectrometry

Reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC) method using two 15cm sub–2μm particles octadecylsilica gel columns is developed with the goal to separate and unambiguously identify a large number of lipid species in biological samples. The identification is performed by the coupling with high-resolution tandem mass spectrometry (MS/MS) using quadrupole – time-of-flight (QTOF) instrument. Electrospray ionization (ESI) full scan and tandem mass spectra are measured in both polarity modes with the mass accuracy better than 5ppm, which provides a high confidence of lipid identification. Over 400 lipid species covering 14 polar and nonpolar lipid classes from 5 lipid categories are identified in total lipid extracts of human plasma, human urine and porcine brain. The general dependences of relative retention times on relative carbon number or relative double bond number are constructed and fit with the second degree polynomial regression. The regular retention patterns in homologous lipid series provide additional identification point for UHPLC/MS lipidomic analysis, which increases the confidence of lipid identification. The reprocessing of previously published data by our and other groups measured in the RP mode and ultrahigh-performance supercritical fluid chromatography on the silica column shows more generic applicability of the polynomial regression for the description of retention behavior and the prediction of retention times. The novelty of this work is the characterization of general trends in the retention behavior of lipids within logical series with constant fatty acyl length or double bond number, which may be used as an additional criterion to increase the confidence of lipid identification.

Hydrophilic interaction liquid chromatography⿿mass spectrometry of (lyso)phosphatidic acids, (lyso)phosphatidylserines and other lipid classes

The goal of this work is a systematic optimization of hydrophilic interaction liquid chromatography (HILIC) separation of acidic lipid classes (namely phosphatidic acids⿿PA, lysophosphatidic acids⿿LPA, phosphatidylserines⿿PS and lysophosphatidylserines⿿LPS) and other lipid classes under mass spectrometry (MS) compatible conditions. The main parameters included in this optimization are the type of stationary phases used in HILIC, pH of the mobile phase, the type and concentration of mobile phase additives. Nine HILIC columns with different chemistries (unmodified silica, modified silica using diol, 2-picolylamine, diethylamine and 1-aminoanthracene and hydride silica) are compared with the emphasis on peak shapes of acidic lipid classes. The optimization of pH is correlated with the theoretical calculation of acidobasic equilibria of studied lipid classes. The final method using the hydride column, pH 4 adjusted by formic acid and the gradient of acetonitrile and 40mmol/L of aqueous ammonium formate provides good peak shapes for all analyzed lipid classes including acidic lipids. This method is applied for the identification of lipids in real samples of porcine brain and kidney extracts.

High-Throughput and Comprehensive Lipidomic Analysis Using Ultrahigh-Performance Supercritical Fluid Chromatography-Mass Spectrometry.

New analytical approach for high-throughput and comprehensive lipidomic analysis of biological samples using ultrahigh-performance supercritical fluid chromatography (UHPSFC) with electrospray ionization-mass spectrometry (ESI-MS) is presented in this work as an alternative approach to established shotgun MS or high-performance liquid chromatography-MS. The lipid class separation is performed by UHPSFC method based on 1.7 μm particle-bridged ethylene hybrid silica column with a gradient of methanol-water-ammonium acetate mixture as a modifier. All parameters of UHPSFC conditions are carefully optimized and their influence on the chromatographic behavior of lipids is discussed. The final UHPSFC/ESI-MS method enables a fast separation of 30 nonpolar and polar lipid classes within 6 min analysis covering 6 main lipid categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. Individual lipid species within lipid classes are identified based on positive and negative-ion full-scan and tandem mass spectra measured with high mass accuracy and high resolving power. Developed UHPSFC/ESI-MS method is applied for the analysis of porcine brain extract as a complex lipidomic sample, where 24 lipid classes containing 436 lipid species are identified. The method is validated for the quantitative analysis of lipid species in biological tissues using internal standards for each lipid class. This high-throughput, comprehensive and accurate UHPSFC/ESI-MS method is suitable for the lipidomic analysis of large sample sets in the clinical research.

Identification of synaptosomal proteins binding to monomeric and oligomeric α-synuclein.

Monomeric α-synuclein (αSN) species are abundant in nerve terminals where they are hypothesized to play a physiological role related to synaptic vesicle turn-over. In Parkinson’s disease (PD) and dementia with Lewy body (DLB), αSN accumulates as aggregated soluble oligomers in terminals, axons and the somatodendritic compartment and insoluble filaments in Lewy inclusions and Lewy neurites. The autosomal dominant heritability associated to mutations in the αSN gene suggest a gain of function associated to aggregated αSN. We have conducted a proteomic screen to identify the αSN interactome in brain synaptosomes. Porcine brain synaptosomes were fractionated, solubilized in non-denaturing detergent and subjected to co-immunoprecipitation using purified recombinant human αSN monomers or oligomers as bait. The isolated αSN binding proteins were identified with LC-LTQ-orbitrap tandem mass spectrometry and quantified by peak area using Windows client application, Skyline Targeted Proteomic Environment. Data are available via ProteomeXchange with identifier PXD001462. To quantify the preferential binding an average fold increase was calculated by comparing binding to monomer and oligomer. We identified 10 proteins preferentially binding monomer, and 76 binding preferentially to oligomer and a group of 92 proteins not displaying any preferred conformation of αSN. The proteomic data were validated by immunoprecipitation in both human and porcine brain extracts using antibodies against monomer αSN interactors: Abl interactor 1, and myelin proteolipid protein, and oligomer interactors: glutamate decarboxylase 2, synapsin 1, glial fibrillary acidic protein, and VAMP-2. We demonstrate the existence of αSN conformation selective ligands and present lists of proteins, whose identity and functions will be useful for modeling normal and pathological αSN dependent processes.

Neuroprotective Effect against Alzheimer's Disease of Porcine Brain Extract

Problem statement: Despite the increasing importance of Alzheimer’s disease, no effective therapeutic strategy is available. Therefore, neuroprotective strategy is still required. Recent findings show that numerous substances possessing antioxidant can improve neurodegeneration and memory impairment. Based on the antioxidant effect and its reputation to serve as brain tonic in traditional folklore, we hypothesized that porcine brain extract could mitigate neurodegeneration and memory impairment. Therefore, this study was set up to determine the effect of porcine brain extract on memory impairment and neurodegeneration in animal models of Alzheimer’s disease. Approach: Male Wistar rats (180-220 g) had been orally given porcine brain extract at doses of 0.5 and 2.5 mg kg-1 BW for a period of 4 weeks before and 1 week after the induction of cognitive deficit condition as those found in early phase of Alzheimer’s disease via the intraventricular injection of AF64A, a cholinotoxin. Rats were assessed the spatial memory using Morris water maze test. Then, they were determined neuron density in hippocampus using histological techniques. Moreover, the assessment of acetylcholinesterase (AChE) activity and malondialdehyde (MDA) level in hippocampus were also performed. Results: It was found that both doses of porcine brain extract could enhance memory, neuron and cholinergic neuron density in all subregions of hippocampus. In addition, the decreased AChE and MDA were also observed. Therefore, our results suggested that the possible underlying mechanism of the extract might occur partly via the decrease in oxidative stress marker, MDA and AChE. Conclusion: This study clearly demonstrates that porcine brain extract can protect against memory impairment and neurodegeneration in animal model of Alzheimer’s disease. Therefore, it should be serve as the potential food supplement or adjuvant therapy against Alzheimer’s disease and other age-related cognitive decline. However, further researches about therapeutic range and possible active ingredient are necessary.

Time-Dependent Oxidation during Nano-Assisted Laser Desorption Ionization Mass Spectrometry: A Useful Tool for Structure Determination or a Source of Possible Confusion?

This work reports on a new and extremely simple approach for determination of a double bond position by a laser desorption ionization mass spectrometry. It is solely based on the catalytic properties of nanostructured surfaces used in nanoassisted laser desorption ionization experiments. These surfaces can induce oxidation of analytes, which results in a mass shift that can be detected by mass spectrometry. If a site of unsaturation is oxidized and cleaved, the m/z difference is diagnostic of the position of a double bond. By demonstrating that the oxidation depends on the analyte surface dwell time, it was proven that it is caused by the surface activity and not by the laser desorption ionization process itself. Control matrix-assisted laser desorption/ionization (MALDI) experiment showed only a limited partial oxidation and no time dependency of the process. The ability to determine a position of a double bond was demonstrated on polyunsaturated phospholipids and cyclosporine A. In some other cases, however, the unexpected oxidation could cause confusion, as demonstrated for a glycosphingolipid from a porcine brain extract.

High Accuracy Mass Measurement Approach in the Identification of Phospholipids in Lipid Extracts: 7 T Fourier-transform Mass Spectrometry and MS/MS Validation

Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305-764, KoreaReceived September 29, 2010, Accepted February 1, 2011In the present study, the approach of high accuracy mass measurements for phospholipid identifications wasevaluated using a 7 T ESI-FTMS/linear ion trap MS/MS. Experiments were carried out for porcine brain,bovine liver, and soybean total lipid extracts in both positive and negative ion modes. In total, 59, 55, and 18phospholipid species were characterized in the positive ion mode for porcine brain, bovine liver, and soybeanlipid extracts, respectively. Assigned lipid classes were PC, PE, PEt, PS, and SM. In the negative ion mode,PG, PS, PA, PE, and PI classes were observed. In the negative ion mode, for porcine brain, bovine liver, andsoybean lipid extracts, 28, 34, and 29 species were characterized, respectively. Comparison of our results withthose obtained by other groups using derivatization-LC-APCI MS and nano-RP-LC-MS/MS showed that ourapproach can characterize PC species as effectively as those methods could. In conclusion, we demonstratedthat high accuracy mass measurements of total lipid extracts using a high resolution FTMS, particularly, 7TFTMS, plus ion-trap MS/MS are very useful in profiling lipid compositions in biological samples. Abbreviation: PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PEt, phosphatid-ylethanol; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphtidylserine; SM, sphingomyelinKey Words : Lipids, Fourier transform ion cyclotron resonance mass spectrometer, Linear ion trap, Accuratemass measurements, Lipid databaseIntroductionFourier transform-ion cyclotron resonance mass spectro-metry (FT-ICR MS or FTMS) offers an ultrahigh massresolution and extraordinary mass accuracy, which has thusled to its wide applications in protein/peptide analysis.

Current biochemistry, molecular biology, and clinical relevance of natriuretic peptides

The mammalian natriuretic peptide family consists of atrial (ANP), brain [B-type; BNP] and C-type natriuretic peptide (CNP) and three receptors, natriuretic receptors-A (NPR-A), -B (NPR-B) and -C (NPR-C). Both ANP and BNP are abundantly expressed in the heart and are secreted mainly from the atria and ventricles, respectively. By contrast, CNP is mainly expressed in the central nervous system, bone and vasculature. Plasma concentrations of both ANP and BNP are elevated in patients with cardiovascular disease, though the magnitude of the increase in BNP is usually greater than the increase in ANP. This makes BNP is a clinically useful diagnostic marker for several pathophysiological conditions, including heart failure, ventricular remodeling and pulmonary hypertension, among others. Recent studies have shown that in addition to BNP-32, proBNP-108 also circulates in human plasma and that levels of both forms are increased in heart failure. Furthermore, proBNP-108 is O-glycosylated and circulates at higher levels in patients with severe heart failure. In this review we discuss recent progress in our understanding of the biochemistry, molecular biology and clinical relevance of the natriuretic peptide system.

Porcine Brain Extract Attenuates Memory Impairments Induced by Focal Cerebral Ischemia

Problem statement: Stroke or cerebral ischemia has been recognized as one important problem worldwide. To date, the effectiveness of protective and therapeutic strategies against stroke is still very limited. Therefore, the development of novel strategy is required. Porcine brain is traditional believed to improve brain functions. Recent studies showed that the extract of porcine brain could protect against brain damage related to the oxidative stress, therefore, we hypothesized that it could protect against brain damage in stroke. Approach: To test the potential of porcine brain extract as the novel protective supplement against stroke, various doses of porcine brain extract at doses of 0.5 and 2.5 mg kg-1 b.w. were orally given to male Wistar rats, weighing 300-350 g, at the period of 14 days before and 21 days after the occlusion of right middle cerebral artery. Then, all rats were determined the neurological score, motor performance, cognitive function and brain infarct volume. Moreover, the possible neuroprotective mechanisms of the extract were also determined via the alteration of Malondialdehyde (MDA) or lipid peroxidation product and via the activities of scavenger enzymes including Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione Peroxides (GPx). Results: The results showed that the low dose of porcine extract decreased the infarct volume and improved brain functions including neurological score, motor performance and memory deficit. In addition, it also decreased MDA but increased the activities of SOD, CAT and GPx. Conclusion: Our results suggested the potential role of porcine brain extract as neuroprotectant. The possible underlying mechanism appeared to be related to the enhanced activities of SOD, CAT and GPx which in turn resulted in the decrease MDA. Moreover, our findings may shed light on the pharmacologic basis for the clinical application of traditional Chinese medicine to protect against stroke.

Targeted analysis of ganglioside and sulfatide molecular species by LC/ESI-MS/MS with theoretically expanded multiple reaction monitoring

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has been applied primarily to the analysis of glycosphingolipids separated from other complex mixtures by TLC, but it is difficult to obtain quantitative profiling of each glycosphingolipid among the different spots on TLC by MALDI-MS. Thus, the development of a convenient approach that utilizes liquid chromatography/electrospray ionization (LC/ESI)-MS has received interest. However, previously reported methods have been insufficient to separate and distinguish each ganglioside class. Here we report an effective method for the targeted analysis of theoretically expected ganglioside molecular species by LC/ESI tandem mass spectrometry (LC/ESI-MS/MS) in combination with multiple reaction monitoring (MRM). MRM detection specific for sialic acid enabled us to analyze ganglioside standards such as GM1, GM2, GM3, GD1, and GT1 at picomolar to femtomolar levels. Furthermore, other gangliosides, such as GD2, GD3, GT2, GT3, and GQ1, were also detected in glycosphingolipid standard mixtures from porcine brain and acidic glycolipid extract from mouse brain by theoretically expanded MRM. We found that this approach was also applicable to sulfatides contained in the glycosphingolipid mixtures. In addition, we established a method to separate and distinguish regioisomeric gangliosides, such as GM1a and -1b, GD1a, -1b, and -1c, and GT1a, -1b, and -1c with diagnostic sugar chains in the MRM.

Effect of porcine brain growth factor on primary cell cultures and BHK-21 [C-13] cell line

Growth factors from neural tissues have been described as potent mitogens for a wide variety of mesoderm- and ectoderm-derived cells in vitro. We used porcine brain extract for in vitro testing of proliferation properties on primary ovarian cells, uterine cells, and cardiomyocytes in culture as well as for BHK-21 [C-13] cell line. The addition of this extract accelerates proliferation in all examined cultures. It also lowers serum requirement and shortens the cultivation period for BHK-21 [C-13] cells. Fibroblast growth factors from brain of different species, but not porcine, are already characterized and their proliferative effect proved. Therefore, we purified, determined, and confirmed the presence of basic fibroblast growth factor in porcine brain extract by Western blot analysis and showed its biological activity on BHK-21 [C-13] cells.

Identification of Phospholipid Molecular Species in Porcine Brain Extracts Using High Mass Accuracy of 4.7 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Lipids play a critical role in many metabolic and biological processes, including energy production and storage, the formation and functioning of cellular membranes, and signal transduction. The biological importance of lipids has drawn extensive attention to the analysis of lipid molecular species. In particular, mass spectrometry (MS) has been shown to be a powerful tool in elucidating the diversity of lipid species and their participation in regulating cellular functions. In recent years, Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) has been increasingly utilized, particularly in profiling global lipid distribution. Its unprecedented mass accuracy enables the discernment of mass differences of less than 0.01 mass unit, such as those often observed in lipid analyses. Under the assumption that the cation species are known, the minimum accuracy for assigning the chemical composition of lipid species is 21.38 ppm. With the current FTICR-MS technology, this mass accuracy is achievable even in a broad band mass analysis in the range m/z 400-1,000. In addition, databases(DBs) with extensive entries are now available and play an essential role in identifying individual lipid molecular species among an enormous number of lipids. For example, the group of Taguchi has demonstrated that low magnetic field (4.7 T) FTICR MS can be applied to identify the molecular species of phophatidylethanolamine (PE) of Caenorhabditis elegans and their oxidized metabolites. Electrospray ionization (ESI) FTICR-MS of total lipid extracts has also been shown to be useful in detecting differences between the distributions of highly heterogeneous mixtures of lipids found in eukaryotic cells. In the present study, 4.7 T ESI-FTICR-MS was performed on porcine brain extracts to demonstrate its lipid profiling capability, and these results were rigorously checked with ESI-MS/MS using a linear ion-trap mass spectrometer. To our knowledge, this is the first study to rigorously validate the preliminary assignments of lipid species based on high accuracy mass values of FTICR MS by comparison with MS/MS results. In addition, a recently publicized web DB, “Lipidsearch”, with extensive lipid entries (advertised to have more than 100,000 entries) is fully utilized to obtain a lipid profile of the porcine brain sample.

Invited commentary

Since its identification in porcine brain extract, interest in C-type natriuretic peptide (CNP) has continuously increased because of its unexpected properties. Together with atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), CNP is a member of the natriuretic peptide family that plays an important role in the regulation of diuresis/natriuresis and vascular tone [1], and similar to them it has been recently discovered that it also plays a role in cardiovascular regulation and heart function.