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JoVE Journal
Biochemistry
Quantitative Analyse des Zelllipidoms von Saccharomyces Cerevisiae mit Flüssigchromatogr...
Quantitative Analyse des Zelllipidoms von Saccharomyces Cerevisiae mit Flüssigchromatogr...
JoVE Journal
Biochemistry
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JoVE Journal Biochemistry
Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Quantitative Analyse des Zelllipidoms von Saccharomyces Cerevisiae mit Flüssigchromatographie gekoppelt mit Tandem-Massenspektrometrie

Full Text
7,929 Views
08:56 min
March 8, 2020

DOI: 10.3791/60616-v

Karamat Mohammad1, Heng Jiang2, Md. Israil Hossain1, Vladimir I. Titorenko1

1Department of Biology,Concordia University, 2Department of Chemistry and Biochemistry, Centre for Biological Applications of Mass Spectrometry,Concordia University

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Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This protocol provides a robust and sensitive method for identifying and quantifying major lipid classes in Saccharomyces cerevisiae using liquid chromatography coupled with tandem mass spectrometry. The method allows for the detection of isobaric and isomeric lipidic species.

Key Study Components

Area of Science

  • Neuroscience
  • Biochemistry
  • Cell Biology

Background

  • Lipids play crucial roles in cellular functions and membrane structure.
  • Quantifying lipid classes is essential for understanding metabolic processes.
  • Traditional methods may lack sensitivity or specificity.
  • This protocol aims to improve lipid analysis in yeast models.

Purpose of Study

  • To develop a reliable method for lipid quantification in yeast.
  • To facilitate the study of lipid metabolism in Saccharomyces cerevisiae.
  • To enable the detection of diverse lipid species.

Methods Used

  • Liquid chromatography coupled with tandem mass spectrometry (LC-MS).
  • Single extraction method for lipid isolation.
  • Culture of yeast cells in a controlled environment.
  • Centrifugation for cell collection and lipid extraction.

Main Results

  • Successful quantification of ten different lipid classes.
  • Detection of isobaric and isomeric lipid species.
  • Method demonstrated reproducibility and accuracy.
  • Protocol can be adapted for various experimental setups.

Conclusions

  • The developed method is versatile and sensitive for lipid analysis.
  • It provides a valuable tool for researchers studying lipid metabolism.
  • Further applications may extend to other yeast species and conditions.

Frequently Asked Questions

What is the main advantage of this lipid quantification method?
The method is robust, sensitive, and allows for the detection of diverse lipid species.
How many lipid classes can be quantified using this protocol?
The protocol allows for the quantification of ten different lipid classes.
What type of chromatography is used in this study?
Liquid chromatography coupled with tandem mass spectrometry (LC-MS) is used.
Can this method be applied to other organisms?
While this study focuses on Saccharomyces cerevisiae, the method may be adaptable to other organisms.
What conditions are required for culturing the yeast?
Yeast should be cultured in YNB medium with a final glucose concentration of 2% at 30 degrees Celsius.
Is the method reproducible?
Yes, the method has been shown to be accurate and reproducible.

Wir präsentieren ein Protokoll mit Flüssigchromatographie gekoppelt mit Tandem-Massenspektrometrie, um wichtige zelluläre Lipide in Saccharomyces cerevisiaezu identifizieren und zu quantifizieren. Die beschriebene Methode zur quantitativen Bewertung wichtiger Lipidklassen innerhalb einer Hefezelle ist vielseitig, robust und empfindlich.

Dieses Protokoll bietet eine genaue und reproduzierbare Methode zur Quantifizierung von zehn verschiedenen Lipidklassen von Saccharomyces cerevisiae mit einer einzigen Extraktionsmethode und einer einzigen LC-MS-Methode. Diese Methode ermöglicht den Nachweis und die Quantifizierung von isobarischen und isomerischen Lipidarten mit unterschiedlichen chemischen und physikalischen Eigenschaften. Für Saccharomyces cerevisiae Kultur, fügen Sie zuerst 5 ml sterile 20%Glukose-Stammlösung zu jedem von zwei Erlenmeyer-Flaschen mit sterilem YNB-Medium bis zu einer Endkonzentration von 2%Glukose.

Dann verwenden Sie eine sterile Pipette, um ein Volumen einer über Nacht Hefekultur mit fünf mal zehn bis siebten Hefezellen in jeden Kolben und Kultur die Kolben für mindestens 24 Stunden bei 30 Grad Celsius mit Rotationsschütteln bei 200 Umdrehungen pro Minute zu übertragen. Für die Lipidextraktion bestimmen Sie die Gesamtzahl der Hefezellen pro Milliliter Kultur und übertragen sie fünfmal zehn in die siebte Zelle in ein 15 ml Zentrifugenrohr. Fügen Sie eiskaltes nanoreines Wasser hinzu, um das Gesamtvolumen auf bis zu 15 ml zu erhöhen und die Zellen durch Zentrifugation zu sammeln.

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Biochemie Ausgabe 157 Lipide zelluläres Lipidom unesterifizierte (freie) Fettsäuren Glycerophospholipide neutrale Lipide Sphingolipide Ceramide Kardiolipine Lipidextraktion Lipidomik Flüssigchromatographie Massenspektrometrie

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