Name: quantitative and qualitative method for sphingomyelin by lc-ms using two stable isotopically labeled sphingomyelin species
Uploaded: 2018-05-07T13:00:00.000+00:00
Duration: 8 min 53 s
Description: Watch this Scientific Journal Video about Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species at JoVE.com

كان يؤيد هذا العمل بمنحه بحثية من وزارة التعليم، الثقافة والرياضة، والعلوم والتكنولوجيا من اليابان (كاكينهي) إلى ك. أ. (#15 ك 01691)، المجلس (#15 ك 08625)، (#26461532)، والحصول على منحة لدراسة "مشروع الأمراض المستعصية" من وزارة الصحة والعمل والرعاية الاجتماعية (#201510032A). ونحن نشكر مجموعة ادانز (www.edanzediting.com/ac) لتحرير مسودة لهذه المخطوطة.

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H., Sullards, M. C., Allegood, J. C., Kelly, S., Wang, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingolipidomics:+high-throughput,+structure-specific,+and+quantitative+analysis+of+sphingolipids+by+liquid+chromatography+tandem+mass+spectrometry.">Sphingolipidomics: high-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry.</a> Methods. 36 (2), 207-224 (2005).</li><li>Houjou, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+selective+identification+of+molecular+species+in+phosphatidylcholine+and+sphingomyelin+by+conditional+neutral+loss+scanning+and+MS3.">Rapid and selective identification of molecular species in phosphatidylcholine and sphingomyelin by conditional neutral loss scanning and MS3.</a> Rapid Commun Mass Spectrom. 18 (24), 3123-3130 (2004).</li><li>Hama, K., Fujiwara, Y., Tabata, H., Takahashi, H., Yokoyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+Quantitation+Using+Two+Stable+Isotopically+Labeled+Species+and+Direct+Detection+of+N-Acyl+Moiety+of+Sphingomyelin.">Comprehensive Quantitation Using Two Stable Isotopically Labeled Species and Direct Detection of N-Acyl Moiety of Sphingomyelin.</a> Lipids. , (2017).</li><li>Bligh, E. G., Dyer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+of+total+lipid+extraction+and+purification.">A rapid method of total lipid extraction and purification.</a> Can. J. Biochem. Physiol. 37, 911-917 (1959).</li><li>Gu, H., Liu, G., Wang, J., Aubry, A. F., Arnold, M. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+the+isolation+and+purification+of+total+lipides+from+animal+tissues.">A simple method for the isolation and purification of total lipides from animal tissues.</a> J Biol Chem. 226 (1), 497-509 (1957).</li><li>Thomas, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ozone-induced+dissociation:+elucidation+of+double+bond+position+within+mass-selected+lipid+ions.">Ozone-induced dissociation: elucidation of double bond position within mass-selected lipid ions.</a> Anal Chem. 80 (1), 303-311 (2008).</li><li>Baba, T., Campbell, J. L., Le Blanc, J. C., Baker, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-depth+sphingomyelin+characterization+using+electron+impact+excitation+of+ions+from+organics+and+mass+spectrometry.">In-depth sphingomyelin characterization using electron impact excitation of ions from organics and mass spectrometry.</a> J Lipid Res. 57 (5), 858-867 (2016).</li><li>Ryan, E., Nguyen, C. Q. N., Shiea, C., Reid, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+Structural+Characterization+of+Sphingolipids+via+193+nm+Ultraviolet+Photodissociation+and+Ultra+High+Resolution+Tandem+Mass+Spectrometry.">Detailed Structural Characterization of Sphingolipids via 193 nm Ultraviolet Photodissociation and Ultra High Resolution Tandem Mass Spectrometry.</a> J Am Soc Mass Spectrom. , (2017).</li><li>Pham, H. T., Ly, T., Trevitt, A. J., Mitchell, T. W., Blanksby, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+complex+lipid+isomers+by+radical-directed+dissociation+mass+spectrometry.">Differentiation of complex lipid isomers by radical-directed dissociation mass spectrometry.</a> Anal Chem. 84 (17), 7525-7532 (2012).</li></ol>

الكتاب يعلن أن أي تعارض في المصالح.

في هذا الأسلوب النوعي، حصلنا على مرض التصلب العصبي المتعدد3 أيونات المنتج توافق آراء ساو باولو و نون-مجموعة الأسيل. من الأهمية بمكان لتعيين بشكل صحيح توافق آراء ساو باولو و نون-مجموعة الأسيل. وتحقيقا لهذه الغاية، تجدر الإشارة إلى أن يمكن أيضا الكشف عن الجزيئات الأخرى التي ت...

سفينجوميلين (SM) سفينجوليبيد مشتركة في خلايا الثدييات. SM هو المركب إينتراسيلولارلي1 والحاضر تمهيدا لأخرى سفينجوليبيدس مثل سفينجوسيني-1-فوسفات، والسيراميد، لها أدوار حاسمة في جهاز المناعة الخليوي الاتجار والجلد حاجز التوازن، على التوالي2، 3. وبالتالي، من المهم تحليل دقيق من الأيض SM لتوضيح أدوار سفينجوليبيدس الفسيولوجية والمرضية.
يتكون SM السيراميد وفوسفوريلتشوليني مرتبط بمجموعة السيراميد، وهي كذلك مؤلفة من سفينجوسيني و ن1-هيدروكسي-مجموعة الأسيل. مجموعة متنوعة في أرقام الكربون والرابطة المزدوجة في كل من سفينجوسيني و نون-مجموعة الأسيل ينتج الأنواع عدد السيراميد (و SM). وقد مكن التقدم الذي أحرز مؤخرا في LC-ESI-MS/MS التحليل الكمي والنوعي من خ4،5. في التحليل النوعي، حددت عدد الكربون وسندات مزدوجة من سفينجويد "لكب ن خ" تعيين المنتج أطياف أيون لكب. ومع ذلك، المعلومات الهيكلية من N-اشيل moiety لم يتم مباشرة الحصول عليه لأنه لم يبلغ الأيونات المنتج المطابق، وذلك ن-اشيل مويتيس تم استخلاصه بتحليل تفاضلي بين أيونات السلائف و المنتج أيونات تناظر لكب في4،وسائط أيون إيجابية وسلبية على حد سواء5. وفي هذا التقرير، نقدم وسيلة للكشف عن الأيونات المنتج لكب و نون-اشيل moiety في نفس الوقت في وضع3 مرض التصلب العصبي المتعدد باستخدام الرباعي الثلاثي والرباعي الخطي أيون فخ الكتلي، مما يسهل الهيكلية الدقيقة المضاربة من كل الأنواع خ6.
أيون قمع (أو تعزيز) الآثار الناجمة عن المصفوفة في عينات بيولوجية تعوق القياس الكمي الدقيق في تحليل LC-أي إس أي--مرض التصلب العصبي المتعدد، وذلك، من المستصوب إنشاء منحنيات المعايرة لجميع التحاليل للفائدة في مصفوفة متماثلة العينة البيولوجية. بيد أن هذه الاستراتيجية غير مجدية لأنه يكاد يكون من المستحيل أن تعد جميع الأنواع SM في العينات البيولوجية، لا سيما في تحليل شامل. وبالتالي، فمن العملي لإنشاء منحنى معايرة وتحديد النطاق الكمي باستخدام أنواع ممثل SM ارتفعت في العينات البيولوجية. استخدمنا نوعين من أنواع SM المسمى نظير لإنشاء منحنى معايرة؛ واحد كان يستخدم لمعيار داخلية والآخر لمعيار مركب. أننا نكتشف كمية صغيرة من الأنواع المسماة نظير SM كمجمع قياسية بنجاح الحصول على منحنى معايرة و تتراوح الكمية6وارتفعت في العينات البيولوجية.

<table><tbody><tr><td>PBS</td><td>ThermoFisher</td><td>10010023</td><td></td></tr><tr><td>100 mm tissue culture dish</td><td>IWAKI</td><td>3020-100</td><td></td></tr><tr><td>Cell scraper</td><td>IWAKI</td><td>9000-220</td><td></td></tr><tr><td>Siliconized 2.0 mL tube</td><td>Fisher Scientific</td><td>02-681-321</td><td></td></tr><tr><td>Test tube</td><td>IWAKI</td><td>TST SCR 16-100</td><td></td></tr><tr><td>Teflon-lined screw cap</td><td>IWAKI</td><td>9998CAP415-15</td><td></td></tr><tr><td>Disposable glass tube</td><td>IWAKI</td><td>9832-1310</td><td></td></tr><tr><td>CAPCELL PAK C18 ACR 3 &amp;micro;m 1.5 mm I.D. x 100 mm</td><td>Shiseido</td><td>92223</td><td>Guard cartridge is inserted into cartridge holder, and linked to C18 column</td></tr><tr><td>CAPCELL C18 MGII S-3 2.0 mm x 10 mm GUARD CARTRIDGE</td><td>Shiseido</td><td>12197</td><td></td></tr><tr><td>Cartridge holder</td><td>Shiseido</td><td>12415</td><td></td></tr><tr><td>Acetonitrile</td><td>Wako</td><td>018-19853</td><td></td></tr><tr><td>2-Propanol (Isopropyl Alcohol)</td><td>Wako</td><td>161-09163</td><td></td></tr><tr><td>Methanol</td><td>Wako</td><td>134-14523</td><td></td></tr><tr><td>Formic acid</td><td>Wako</td><td>066-00466</td><td></td></tr><tr><td>28% Ammonia water</td><td>Wako</td><td>016-03146</td><td></td></tr><tr><td>Sonicator (bath type)</td><td>SHARP</td><td>UT-206H</td><td></td></tr><tr><td>Vortex mixer for glass test tube</td><td>TAITEC</td><td>Mix-EVR</td><td></td></tr><tr><td>1.4 mL glass vial</td><td>Tomsic</td><td>500-1982</td><td>Samples are stored in 1.4 mL glass vial sealed with screw cap and 8 mm septum at -20&amp;deg;C</td></tr><tr><td>8 mm septum</td><td>Tomsic</td><td>200-3322</td><td>When samples are analyzed, screw caps are replaced with screw caps with slit septum</td></tr><tr><td>Screw cap for 1.4 mm glass vial</td><td>Tomsic</td><td>500-2762</td><td></td></tr><tr><td>Screw cap with slit septum</td><td>Shimadzu GLC</td><td>GLCTV-803</td><td></td></tr><tr><td>PVDF 0.22 &amp;micro;m filter</td><td>Millipore</td><td>SLGVR04NL</td><td></td></tr><tr><td>Triple quadrupole and quadrupole linear ion trap mass spectrometry</td><td>SCIEX</td><td>QTRAP4500</td><td></td></tr><tr><td>The software for data acquisition and analysis of product ion spectra</td><td>SCIEX</td><td>Analyst</td><td></td></tr><tr><td>The software for data integration in quantitative analysis</td><td>SCIEX</td><td>MultiQuant</td><td></td></tr><tr><td>HPLC system</td><td>Shimadzu</td><td>Nexera</td><td></td></tr><tr><td>Glass bottle</td><td>Sansyo</td><td>85-0002</td><td></td></tr><tr><td>d18:1/24:0 sphingomyelin</td><td>Avanti Polar Lipids</td><td>860592P</td><td></td></tr><tr><td>Sphingosylphosphorylcholine</td><td>Merck</td><td>567735</td><td></td></tr><tr><td>Fetal bovine serum</td><td>ThermoFisher&amp;nbsp;</td><td>26140079</td><td></td></tr><tr><td>L-glutamine</td><td>ThermoFisher&amp;nbsp;</td><td>25030081</td><td></td></tr><tr><td>Penicillin and streptomycin</td><td>Sigma</td><td>P4333</td><td></td></tr><tr><td>Eagle&amp;rsquo;s minimum essential medium</td><td>Sigma</td><td>M4655</td><td></td></tr></tbody></table>

quantitative and qualitative method for sphingomyelin by lc-ms using two stable isotopically labeled sphingomyelin species

نحن نقدم طريقة تحليل سفينجوميلين (SM) نوعيا وكمياً عن طريق التأين-جنبا إلى جنب كروماتوغرافيا سائلة-اليكتروسبراي الكتلي (LC-ESI-MS/MS). خ سفينجوليبيد مشتركة تتألف من فوسفوريلتشوليني والسيراميد كعنصر ماء ومسعور، على التوالي. عدد من الأنواع SM موجودة في خلايا الثدييات بسبب مجموعة متنوعة في سفينجويد طويلة السلسلة الأساسية (لكب) و نون-اشيل moiety في السيراميد. في هذا التقرير، نعرض طريقة لتقدير عدد الكربون وسندات مزدوجة في لكب و نون-moiety اشيل استناداً على أيونات المنتج المطابق في تجارب MS/MS/MS (3من مرض التصلب العصبي المتعدد). وبالإضافة إلى ذلك، نقدم أسلوب تحليل الكمي ل SM استخدام اثنين مستقرة نظير المسمى SM الأنواع، مما يسهل تحديد النطاق المستخدمة في كوانتيتيشن ن خ. الأسلوب الحالي سيكون مفيداً في وصف مجموعة متنوعة من أنواع SM في العينات البيولوجية والمنتجات الصناعية مثل مستحضرات التجميل.

تصنيعه كيميائيا d18:1/SM (الشكل 1أ) 24:0 و d18:1/24:0 SM في عينات الدهون المستخرجة من خلايا هيلا (الشكل 1ب) تم تحليلها بواسطة LC-أي إس أي--مرض التصلب العصبي المتعدد توظيف3 [م + هكوو]– و [م-CH3]- أيونات السلائف الأولى و...

Watch this Scientific Journal Video about Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species at JoVE.com

Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species

الأسلوب الكمي والنوعي سفينجوميلين من LC-MS استخدام اثنين مستقرة نظير المسمى الأنواع سفينجوميلين

هنا، نحن نقدم بروتوكول للتحديد الكمي وتأهيل كل الأنواع سفينجوميلين باستخدام رصد رد فعل متعددة ووضع MS/MS/MS، على التوالي.

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass ...

quantitative-qualitative-method-for-sphingomyelin-lc-ms-using-two

Research

JoVE Journal

Biochemistry

9.7K Views.  Teikyo University. هنا، نحن نقدم بروتوكول للتحديد الكمي وتأهيل كل الأنواع سفينجوميلين باستخدام رصد رد فعل متعددة ووضع MS/MS/MS، على التوالي.

Video: Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

الخلوية لاستخراج الدهن المستهدفة التخفيف النظائر المستقرة تحليل السائل الطيف اللوني القداس

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Bioengineering

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

الدهون عزل القطرة عن التحليل الكمي الطيف الكتلي

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

Here we show isolated human platelets can be used as an accessible ex vivo model to study metabolic adaptations in response to the complex I inhibitor rotenone. This approach employs isotopic tracing and relative quantification by liquid chromatography-mass spectrometry and can be applied to a variety of study designs.

LC-MS تحليل الصفائح الدموية البشرية باعتبارها منصة لدراسة الميتوكوندريا الأيض

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

Environment

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Radiolabeling والكمي لمستويات الخلايا من Phosphoinositides التي كتبها التلألؤ تدفق عالية الأداء اللوني السائل الديناميكي

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Chemistry

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

تحليل الطيفي الشامل من المستضدات Glycosphingolipid

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

Immunology and Infection

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

التحليل الكمي للدهون الخلوية من Saccharomyces Cerevisiae باستخدام الكروماتوغرافيا السائلة إلى جانب قياس الطيف الكتلي جنبا إلى جنب

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether sphingolipids are altered in a disease and investigate whether sphingolipids can be targeted clinically. However, properly powered prospective studies that acquire tissues directly from the surgical suite can be time consuming, and technically, logistically, and administratively challenging. In contrast, retrospective studies can take advantage of cryopreserved human specimens already available, usually in large numbers, at tissue biorepositories. Other advantages of procuring tissues from biorepositories include access to information associated with the tissue specimens including histology, pathology, and in some instances clinicopathological variables, all of which can be used to examine correlations with lipidomics data. However, technical limitations related to the incompatibility of optimal cutting temperature compound (OCT) used in the cryopreservation and mass spectrometry is a technical barrier for the analysis of lipids. However, we have previously shown that OCT can be easily removed from human biorepository specimens through cycles of washes and centrifugation without altering their sphingolipid content. We have also previously established that sphingolipids in human tissues cryopreserved in OCT are stable for up to 16 years. In this report, we outline the steps and workflow to analyze sphingolipids in human tissue specimens that are embedded in OCT, including washing tissues, weighing tissues for data normalization, the extraction of lipids, preparation of samples for analysis by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), mass spectrometry data integration, data normalization, and data analysis.

Sphingolipids are bioactive metabolites with well-established roles in human disease. Characterizing alterations in tissues with mass-spectrometry can reveal roles in disease etiology or identify therapeutic targets. However, the OCT-compound used for cryopreservation in biorepositories interferes with mass-spectrometry. We outline methods to analyze sphingolipids in human tissues embedded in OCT with LC-ESI-MS/MS.

تحضير الأنسجة البشرية المضمنة في مركب درجة حرارة القطع الأمثل لتحليل مطياف الكتلة

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether ...

Cancer Research

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

والتقييم الكمي لاستخدام الطيف Lipidome الخميرة Electrospray قداس التأين

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Biology

فحص الهياكل الدهنية باستخدام وسم النظائر المستقرة

Tracing de novo Lipids using Stable Isotope Labeling LC-TIMS-TOF MS/MS

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable isotope labeling (SIL) offers several advantages for the study of lipid distribution, mobilization, and metabolism, as well as de novo lipid synthesis. The successful implementation of the SIL technique requires the removal of interferences from endogenous molecules. In the present work, we describe a high-throughput analytical protocol for the screening of SIL lipids from biological samples; examples will be shown of lipid de novo identification during mosquito ovary development. The use of complementary liquid chromatography trapped ion mobility spectrometry and mass spectrometry allows for the separation and lipids assignment from a single sample in a single scan (&lt;1 h). The described approach takes advantage of recent developments in data-dependent acquisition and data-independent acquisition, using parallel accumulation in the mobility trap followed by sequential fragmentation and collision-induced dissociation. The measurement of SIL at the fatty acid chain level reveals changes in lipid dynamics during the ovary development of mosquitoes. The lipids de novo structures are confidently assigned based on their retention time, mobility, and fragmentation pattern.

تسليط الضوء على المؤلف: القياس الكمي للعينات الدهنية المعقدة باستخدام وسم النظائر المستقرة

Presented here is a method for screening lipid structures through stable isotope labeling by using their retention time, mobility, and fragmentation.

تتبع الدهون الجديدة باستخدام وسم النظائر المستقرة LC-TIMS-TOF MS / MS

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable ...

الأسلوب الكمي والنوعي سفينجوميلين من LC-MS استخدام اثنين مستقرة نظير المسمى الأنواع سفينجوميلين

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