title: Characterization of sphingosine-1-phosphate lyase activity by electrospray ionization-liquid chromatography/tandem mass spectrometry quantitation of (2E)-hexadecenal.
abstract: 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.
authors.affiliation: Department of Medicine, University of Illinois at Chicago, 60612, USA. eberdysh@uic.edu
authors.forename: Evgeny V
authors.initials: EV
authors.lastname: Berdyshev
authors.forename: Jonathan
authors.initials: J
authors.lastname: Goya
authors.forename: Irina
authors.initials: I
authors.lastname: Gorshkova
authors.forename: Glenn D
authors.initials: GD
authors.lastname: Prestwich
authors.forename: Hoe-Sup
authors.initials: HS
authors.lastname: Byun
authors.forename: Robert
authors.initials: R
authors.lastname: Bittman
authors.forename: Viswanathan
authors.initials: V
authors.lastname: Natarajan
mesh_terms: 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
chemical_list: 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
references.citation: Curr Med Chem. 2009;16(16):2021-41
references.pmid: 19519379
references.citation: Adv Enzyme Regul. 2010;50(1):349-62
references.pmid: 19914275
references.citation: J Biol Chem. 2005 Oct 7;280(40):33697-700
references.pmid: 16118221
references.citation: Science. 2005 Sep 9;309(5741):1735-9
references.pmid: 16151014
references.citation: Ann Clin Lab Sci. 1977 Nov-Dec;7(6):455-68
references.pmid: 337888
references.citation: Nat Rev Mol Cell Biol. 2008 Feb;9(2):139-50
references.pmid: 18216770
references.citation: Circ Res. 2004 Apr 2;94(6):724-34
references.pmid: 15059942
references.citation: Biochem Biophys Res Commun. 2009 Mar 6;380(2):366-70
references.pmid: 19250638
references.citation: Vet Clin Pathol. 1998;27(3):71-78
references.pmid: 12075541
references.citation: J Biol Chem. 1991 Jul 5;266(19):12502-7
references.pmid: 2061324
references.citation: Biochimie. 2010 Jun;92(6):716-23
references.pmid: 20167244
references.citation: Pharmacol Rev. 2008 Jun;60(2):181-95
references.pmid: 18552276
references.citation: Genes Cells. 2005 Jun;10(6):605-15
references.pmid: 15938718
references.citation: J Lipid Res. 2007 Dec;48(12):2769-78
references.pmid: 17872591