A mass spectrometry-based method for the assay of ceramide synthase substrate specificity

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

The acyl composition of sphingolipids is determined by the specificity of the enzyme ceramide synthase (EC 2.3.1.24). Ceramide contains a long-chain base (LCB) linked to a variety of fatty acids to produce a lipid class with potentially hundreds of structural variants. An optimized procedure for the assay of ceramide synthase in yeast microsomes is reported that uses mass spectrometry to detect any possible LCB and fatty acid combination synthesized from unlabeled substrates provided in the reaction. The assay requires the delivery of substrates with bovine serum albumin for maximum activity within defined limits of substrate concentration and specific methods to stop the reaction and extract the lipid that avoid the non-enzymatic synthesis of ceramide. The activity of ceramide synthase in yeast microsomes is demonstrated with the four natural LCBs found in yeast along with six saturated and two unsaturated fatty acyl-coenzyme As from 16 to 26 carbons in length. The procedure allows for the determination of substrate specificity and kinetic parameters toward natural substrates for ceramide synthase from potentially any organism.

Original languageEnglish (US)
Pages (from-to)96-101
Number of pages6
JournalAnalytical Biochemistry
Volume478
DOIs
StatePublished - Jun 1 2015

Fingerprint

Substrate Specificity
Mass spectrometry
Assays
Mass Spectrometry
Yeasts
Ceramides
Yeast
Substrates
Microsomes
Fatty Acids
Lipids
Sphingolipids
Coenzymes
Bovine Serum Albumin
Carbon
Kinetic parameters
dihydroceramide desaturase
Enzymes
Chemical analysis

Keywords

  • Ceramide synthase
  • Enzyme assay
  • Mass spectrometry
  • Sphingolipids
  • Substrate specificity

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

A mass spectrometry-based method for the assay of ceramide synthase substrate specificity. / Luttgeharm, Kyle D.; Cahoon, Edgar B.; Markham, Jonathan E.

In: Analytical Biochemistry, Vol. 478, 01.06.2015, p. 96-101.

Research output: Contribution to journalArticle

@article{68dcdd7470b14e5197bfa55609ae2beb,
title = "A mass spectrometry-based method for the assay of ceramide synthase substrate specificity",
abstract = "The acyl composition of sphingolipids is determined by the specificity of the enzyme ceramide synthase (EC 2.3.1.24). Ceramide contains a long-chain base (LCB) linked to a variety of fatty acids to produce a lipid class with potentially hundreds of structural variants. An optimized procedure for the assay of ceramide synthase in yeast microsomes is reported that uses mass spectrometry to detect any possible LCB and fatty acid combination synthesized from unlabeled substrates provided in the reaction. The assay requires the delivery of substrates with bovine serum albumin for maximum activity within defined limits of substrate concentration and specific methods to stop the reaction and extract the lipid that avoid the non-enzymatic synthesis of ceramide. The activity of ceramide synthase in yeast microsomes is demonstrated with the four natural LCBs found in yeast along with six saturated and two unsaturated fatty acyl-coenzyme As from 16 to 26 carbons in length. The procedure allows for the determination of substrate specificity and kinetic parameters toward natural substrates for ceramide synthase from potentially any organism.",
keywords = "Ceramide synthase, Enzyme assay, Mass spectrometry, Sphingolipids, Substrate specificity",
author = "Luttgeharm, {Kyle D.} and Cahoon, {Edgar B.} and Markham, {Jonathan E.}",
year = "2015",
month = "6",
day = "1",
doi = "10.1016/j.ab.2015.02.016",
language = "English (US)",
volume = "478",
pages = "96--101",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press Inc.",

}

TY - JOUR

T1 - A mass spectrometry-based method for the assay of ceramide synthase substrate specificity

AU - Luttgeharm, Kyle D.

AU - Cahoon, Edgar B.

AU - Markham, Jonathan E.

PY - 2015/6/1

Y1 - 2015/6/1

N2 - The acyl composition of sphingolipids is determined by the specificity of the enzyme ceramide synthase (EC 2.3.1.24). Ceramide contains a long-chain base (LCB) linked to a variety of fatty acids to produce a lipid class with potentially hundreds of structural variants. An optimized procedure for the assay of ceramide synthase in yeast microsomes is reported that uses mass spectrometry to detect any possible LCB and fatty acid combination synthesized from unlabeled substrates provided in the reaction. The assay requires the delivery of substrates with bovine serum albumin for maximum activity within defined limits of substrate concentration and specific methods to stop the reaction and extract the lipid that avoid the non-enzymatic synthesis of ceramide. The activity of ceramide synthase in yeast microsomes is demonstrated with the four natural LCBs found in yeast along with six saturated and two unsaturated fatty acyl-coenzyme As from 16 to 26 carbons in length. The procedure allows for the determination of substrate specificity and kinetic parameters toward natural substrates for ceramide synthase from potentially any organism.

AB - The acyl composition of sphingolipids is determined by the specificity of the enzyme ceramide synthase (EC 2.3.1.24). Ceramide contains a long-chain base (LCB) linked to a variety of fatty acids to produce a lipid class with potentially hundreds of structural variants. An optimized procedure for the assay of ceramide synthase in yeast microsomes is reported that uses mass spectrometry to detect any possible LCB and fatty acid combination synthesized from unlabeled substrates provided in the reaction. The assay requires the delivery of substrates with bovine serum albumin for maximum activity within defined limits of substrate concentration and specific methods to stop the reaction and extract the lipid that avoid the non-enzymatic synthesis of ceramide. The activity of ceramide synthase in yeast microsomes is demonstrated with the four natural LCBs found in yeast along with six saturated and two unsaturated fatty acyl-coenzyme As from 16 to 26 carbons in length. The procedure allows for the determination of substrate specificity and kinetic parameters toward natural substrates for ceramide synthase from potentially any organism.

KW - Ceramide synthase

KW - Enzyme assay

KW - Mass spectrometry

KW - Sphingolipids

KW - Substrate specificity

UR - http://www.scopus.com/inward/record.url?scp=84926632026&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84926632026&partnerID=8YFLogxK

U2 - 10.1016/j.ab.2015.02.016

DO - 10.1016/j.ab.2015.02.016

M3 - Article

C2 - 25725359

AN - SCOPUS:84926632026

VL - 478

SP - 96

EP - 101

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

ER -