Multiplex analysis of sphingolipids using amine-reactive tags (iTRAQ)

Abstract

This article is available online at http://www.jlr.orgCeramides are central intermediates of sphingolipid biosynthesis and degradation ( 1,2 ). In addition to their metabolic importance, ceramides play a crucial role in a variety of signaling events, including differentiation ( 3 ), senescence ( 4 ), proliferation ( 5 ), and apoptosis ( 6, 7 ). Due to their physiological importance, analysis of their generation as well as their quantifi cation has received considerable attention. Ceramides are quantitatively relatively minor cellular lipid components; thus, highly sensitive detection is required for quantifi cation. Several methods of quantifying ceramide have been reported, including gas chromatography (GC) ( 8 ), high-performance liquid chromatography (HPLC) ( 9-11 ), diacylglycerol kinase assay ( 12 ), and mass spectrometry (MS) ( 13-18 ). Recent advances in MS have made this method a promising approach to the quantitative measurement of ceramides and other lipids. Considering the low cellular concentration of ceramides, it is important to concurrently determine the concentration ratios of ceramides from different samples, such as before and after stimulation of cells. However, simultaneous analysis of ceramide levels across multiple samples has not been reported to date.Isobaric tags for relative and absolute quantitation (iTRAQ) have been developed to compare the protein expression levels in multiple samples ( 19,20 ). These reagents have identical overall mass but vary in terms of the distribution of heavy carbon, nitrogen, and oxygen isotopes within their structure. One big advantage of iTRAQ is enhanced sensitivity because the intensity of the peaks Abstract Ceramides play a crucial role in divergent signaling events, including differentiation, senescence, proliferation, and apoptosis. Ceramides are a minor lipid component in terms of content; thus, highly sensitive detection is required for accurate quantifi cation. The recently developed isobaric tags for relative and absolute quantitation (iTRAQ) method enables a precise comparison of both protein and aminophospholipids. However, iTRAQ tagging had not been applied to the determination of sphingolipids. 31 March 2011. Published, JLR Papers in Press, April 11, 2011 DOI 10.1194 Multiplex analysis of sphingolipids using amine-reactive tags (iTRAQ) Abbreviations: MDCK, Madin Darby canine kidney; MRM, multiple reaction monitoring; PPS, 3-[3-(1,1-bisalkyloxyethyl)pyridine-1-yl] propane-1-sulfonate; SCDase, sphingolipid ceramide N -deacylase; SMase, sphingomyelinase; iTRAQ, isobaric tags for relative and absolute quantitation. Manuscript received 7 February 2011 and in revised form

Widespread tissue distribution and synthetic pathway of polyunsaturated C24:2 sphingolipids in mammals

“…In the present study, we used LC-MS/MS to perform detailed analyses on ceramide species from various mouse tissues. Although PUFA-containing sphingolipids had only been reported in limited tissues [14][15][16][17][18], we found here that C24:2 sphingolipids exist relatively abundantly in a wide range of tissues.…”

“…ceramide is the most limited: there are only a few reports of its existence [13][14][15][16][17]. However, C24:2 ceramide was detected in a wide range of tissues, existing most abundantly in spleen, followed in descending order by small intestine, colon, lung, and liver ( Fig.…”

“…In contrast, the FA moiety of sphingolipids is mainly saturated C16-C24, although monounsaturated sphingolipids with C24:1 also exist ubiquitously [11]. PUFA-containing sphingolipids have only been reported in limited tissues/cells at low levels [13][14][15][16][17]. An exception is spermatozoa (and possibly the surrounding testis), which possesses relatively high amounts of PUFA-containing sphingolipids with ≥C28 [18].…”

Isobaric mass tagging and triple quadrupole mass spectrometry to determine lipid biomarker candidates for Alzheimer's disease

“…However, given the improved throughput, we believe that it would be relatively easy to scale up to several orders of samples to obtain more concrete results. Moreover, it is not necessary to prepare a large number of internal standard substances for this method, correction to QC samples is easy, and rapid quantitative comparison using previously reported isobaric mass tagging method can be achieved [5][6][7][8][9]. It would be ideal to obtain a large-scale pool of QC plasma samples for global QC across all cohort samples.…”

“…Lipids with amino or carboxy groups are the major metabolite groups in the lipidome. As a multiplex method for lipidomics, the analysis of lipids with amino groups such as glycerophosphoethanolamine[5,6], sphingolipids[7], and gangliosides[8] has been already reported. It has been demonstrated to show very good relative quantitation by isobaric mass tagging for amino-phospholipids.…”