High-performance liquid chromatography analysis of molecular species of sphingolipid-related long chain bases and long chain base phosphates in Saccharomyces cerevisiae after derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.
The molecular species of dihydrosphingosines and phytosphingosines and their 1-phosphates with carbon chain lengths from 16 to 20 have been tagged with the fluorescent amino group reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. All these derivatives could be resolved by reversed phase HPLC on a C18 column. A convenient one-pot method is described whereby lipid extracts from strains of Saccharomyces cerevisiae containing carbon chain length homologs of sphingolipid long chain bases and their phosphorylated derivatives were directly reacted with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, ester lipids were deacylated, and the reaction mixtures were subjected to liquid chromatography. Five molecular species of both sphingolipid long chain bases and their phosphorylated derivatives are for the first time separated and analyzed. The procedure is quite sensitive, requiring only approximately 10(8) wild-type cells.
Lester
R L
RL
Department of Molecular and Cellular Biochemistry and Lucille P. Markey Cancer Center, University of Kentucky College of Medicine, 800 Rose Street, Lexington, Kentucky 40536-0298, USA. rlester@uky.edu
Dickson
R C
RC
D000634:Aminoquinolines / Q000032:analysis*; D002219:Carbamates / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D010710:Phosphates / Q000032:analysis*; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013107:Sphingolipids / Q000032:analysis*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000032:analysis
C078178:6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; D000634:Aminoquinolines; D002219:Carbamates; D010710:Phosphates; D013107:Sphingolipids; D013110:Sphingosine