Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers.
Sphingolipids are a highly diverse category of bioactive compounds. This article describes methods that have been validated for the extraction, liquid chromatographic (LC) separation, identification and quantitation of sphingolipids by electrospray ionization, tandem mass spectrometry (ESI-MS/MS) using triple quadrupole (QQQ, API 3000) and quadrupole-linear-ion trap (API 4000 QTrap, operating in QQQ mode) mass spectrometers. Advantages of the QTrap included: greater sensitivity, similar ionization efficiencies for sphingolipids with ceramide versus dihydroceramide backbones, and the ability to identify the ceramide backbone of sphingomyelins using a pseudo-MS3 protocol. Compounds that can be readily quantified using an internal standard cocktail developed by the LIPID MAPS Consortium are: sphingoid bases and sphingoid base 1-phosphates, more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides, and these complex sphingolipids with dihydroceramide backbones. With minor modifications, glucosylceramides and galactosylceramides can be distinguished, and more complex species such as sulfatides can also be quantified, when the internal standards are available. LC ESI-MS/MS can be utilized to quantify a large number of structural and signaling sphingolipids using commercially available internal standards. The application of these methods is illustrated with RAW264.7 cells, a mouse macrophage cell line. These methods should be useful for a wide range of focused (sphingo)lipidomic investigations.
Shaner
Rebecca L
RL
Schools of Chemistry and Biochemistry, Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332-0230, USA
Allegood
Jeremy C
JC
Park
Hyejung
H
Wang
Elaine
E
Kelly
Samuel
S
Haynes
Christopher A
CA
Sullards
M Cameron
MC
Merrill
Alfred H
AH
D000818:Animals; D002457:Cell Extracts / Q000737:chemistry*; D002460:Cell Line; D002518:Ceramides / Q000032:analysis / Q000592:standards; D002853:Chromatography, Liquid / Q000379:methods*; D005227:Fatty Acids / Q000592:standards; D050356:Lipid Metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D011786:Quality Control; D012015:Reference Standards; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D013107:Sphingolipids / Q000032:analysis* / Q000302:isolation & purification / Q000592:standards; D013109:Sphingomyelins / Q000032:analysis / Q000592:standards; D013110:Sphingosine / Q000031:analogs & derivatives / Q000032:analysis / Q000592:standards; D013433:Sulfoglycosphingolipids / Q000032:analysis / Q000592:standards; D053719:Tandem Mass Spectrometry
D002457:Cell Extracts; D002518:Ceramides; D005227:Fatty Acids; D013107:Sphingolipids; D013109:Sphingomyelins; D013433:Sulfoglycosphingolipids; D013110:Sphingosine