Characterization of sphingosine-1-phosphate lyase activity by electrospray ionization-liquid chromatography/tandem mass spectrometry quantitation of (2E)-hexadecenal.
Sphingosine-1-phosphate (S1P) is a sphingolipid signaling molecule crucial for cell survival and proliferation. S1P-mediated signaling is largely controlled through its biosynthesis and degradation, and S1P lyase (S1PL) is the only known enzyme that irreversibly degrades sphingoid base-1-phosphates to phosphoethanolamine and the corresponding fatty aldehydes. S1PL-mediated degradation of S1P results in the formation of (2E)-hexadecenal, whereas hexadecanal is the product of dihydrosphingosine-1-phosphate (DHS1P) degradation. Fatty aldehydes can undergo biotransformation to fatty acids and/or alcohols, making them elusive and rendering the task of fatty aldehyde quantitation challenging. We have developed a simple, highly sensitive, and high-throughput protocol for (2E)-hexadecenal quantitation as a semicarbazone derivative by liquid chromatography-electrospray ionization-tandem mass spectrometry. The approach was applied to determining S1PL activity in vitro with the ability to use as low as 0.25μg of microsomal protein per assay. The method is also applicable to the use of total tissue homogenate as the source of S1PL. A correction for (2E)-hexadecenal disappearance due to its biotransformation during enzymatic reaction is required, especially at higher protein concentrations. The method was applied to confirm FTY720 as the inhibitor of S1PL with an IC₅₀ value of 52.4μM.
Berdyshev
Evgeny V
EV
Department of Medicine, University of Illinois at Chicago, 60612, USA. eberdysh@uic.edu
Goya
Jonathan
J
Gorshkova
Irina
I
Prestwich
Glenn D
GD
Byun
Hoe-Sup
HS
Bittman
Robert
R
Natarajan
Viswanathan
V
D000446:Aldehyde-Lyases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000447:Aldehydes / Q000032:analysis*; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D000068876:Fingolimod Hydrochloride; D006865:Hydrogenation; D007700:Kinetics; D008246:Lysophospholipids / Q000378:metabolism*; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology; D011409:Propylene Glycols / Q000737:chemistry; D051381:Rats; D012664:Semicarbazones / Q000032:analysis; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry
D000447:Aldehydes; D008246:Lysophospholipids; D011409:Propylene Glycols; D012664:Semicarbazones; C060506:sphingosine 1-phosphate; D000446:Aldehyde-Lyases; C022649:sphingosine 1-phosphate lyase (aldolase); D000068876:Fingolimod Hydrochloride; D013110:Sphingosine; C519712:2-hexadecenal
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