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Use of high performance liquid chromatography-electrospray ionization-tandem mass spectrometry for the analysis of ceramide-1-phosphate levels.

ABSTRACT: Ceramide-1-phosphate (C1P) is a bioactive sphingolipid with roles in several biological processes. Currently, high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC ESI-MS/MS) offers the most efficient method of quantifying C1P. However, the published protocols have several drawbacks causing overestimations and carryovers. Here, the reported overestimation of C1P was shown to be due to incomplete neutralization of base hydrolyzed lipid extracts leading to the hydrolysis of SM to C1P. Actual quantity of C1P in cells (6 pmols/10(6) cells) was much lower than previously reported. Also, the major species of C1P produced by ceramide kinase (CERK) was found to be d(18:1/16:0) with a minority of d(18:1/24:1) and d(18:1/24:0). The artifactual production of C1P from SM was used for generating C1Ps as retention time markers. Elimination of carryovers between samples and a 2-fold enhancement in the signal strength was achieved by heating the chromatographic column to 60 (degrees) C. The role of ceramide transport protein (CERT) in supplying substrate to CERK was also revalidated using this new assay. Finally, our results demonstrate the presence of additional pathway(s) for generation of the C1P subspecies, d(18:1/18:0) C1P, as well as a significant portion of d(18:1/16:0), d(18:1/24:1), and d(18:1/24:0). In conclusion, this study introduces a much improved and validated method for detection of C1P by mass spectrometry and demonstrates specific changes in the C1P subspecies profiles upon downregulation of CERK and CERT.

SUBMITTER: Wijesinghe DS

PROVIDER: S-EPMC2817594 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Use of high performance liquid chromatography-electrospray ionization-tandem mass spectrometry for the analysis of ceramide-1-phosphate levels.

Wijesinghe Dayanjan S DS Allegood Jeremy C JC Gentile Luciana B LB Fox Todd E TE Kester Mark M Chalfant Charles E CE

Journal of lipid research 20090804 3

Ceramide-1-phosphate (C1P) is a bioactive sphingolipid with roles in several biological processes. Currently, high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC ESI-MS/MS) offers the most efficient method of quantifying C1P. However, the published protocols have several drawbacks causing overestimations and carryovers. Here, the reported overestimation of C1P was shown to be due to incomplete neutralization of base hydrolyzed lipid extracts leading to t ...[more]

PMID: 19654423

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Project description:We present the first comprehensive capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS) analysis of post-translational modifications derived from H1 and core histones. Using a capillary electrophoresis system equipped with a sheathless high-sensitivity porous sprayer and nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-ESI-MS) as two complementary techniques, we characterized H1 histones isolated from rat testis. Without any pre-separation of the perchloric acid extraction, a total of 70 different modified peptides, including 50 phosphopeptides, were identified in the rat linker histones H1.0, H1a-H1e, and H1t. Out of the 70 modified H1 histone peptides, 27 peptides could be identified with CESI-MS only, and 11 solely with LC-ESI-MS. Immobilized metal-affinity chromatography enrichment prior to MS analysis yielded a total of 55 phosphopeptides; 22 of these peptides could be identified only by CESI-MS, and 19 only by LC-ESI-MS, showing the complementarity of the two techniques. We mapped 42 H1 modification sites, including 31 phosphorylation sites, of which 8 were novel sites. For the analysis of core histones, we chose a different strategy. In a first step, the sulfuric-acid-extracted core histones were pre-separated using reverse-phase high-performance liquid chromatography. Individual rat testis core histone fractions obtained in this way were digested and analyzed via bottom-up CESI-MS. This approach yielded the identification of 42 different modification sites including acetylation (lysine and N(α)-terminal); mono-, di-, and trimethylation; and phosphorylation. When we applied CESI-MS for the analysis of intact core histone subtypes from butyrate-treated mouse tumor cells, we were able to rapidly detect their degree of modification, and we found this method very useful for the separation of isobaric trimethyl and acetyl modifications. Taken together, our results highlight the need for additional techniques for the comprehensive analysis of post-translational modifications. CESI-MS is a promising new proteomics tool as demonstrated by this, the first comprehensive analysis of histone modifications, using rat testis as an example.

Dataset Information

Use of high performance liquid chromatography-electrospray ionization-tandem mass spectrometry for the analysis of ceramide-1-phosphate levels.

Publications

Use of high performance liquid chromatography-electrospray ionization-tandem mass spectrometry for the analysis of ceramide-1-phosphate levels.

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