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Linear ion-trap MSⁿ with high-resolution MS reveals structural diversity of 1-O-acylceramide family in mouse epidermis.

ABSTRACT: 1-O-acylceramide is a new class of epidermal cer-amide (Cer) found in humans and mice. Here, we report an ESI linear ion-trap (LIT) multiple-stage MS (MSⁿ) approach with high resolution toward structural characterization of this lipid family isolated from mice. Molecular species desorbed as the [M + H]⁺ ions were subjected to LIT MS² to yield predominately the [M + H - H₂O]⁺ ions, followed by MS³ to cleave the 1-O-acyl residue to yield the [M + H - H₂O - (1-O-FA)]⁺ ions. The structures of the N-acyl chain and long-chain base (LCB) of the molecule were determined by MS⁴ on [M + H - H₂O - (1-O-FA)]⁺ ions that yielded multiple sets of specific ions. Using this approach, isomers varied in the 1-O-acyl (from 14:0- to 30:0-O-acyl) and N-acyl chains (from 14:0- to 34:1-N-acyl) with 18:1-sphingosine as the major LCB were found for the entire family. Minor isomers consisting of 16:1-, 17:1-, 18:2-, and 19:1-sphingosine LCBs with odd fatty acyl chain or with monounsaturated N- or O-fatty acyl substituents were also identified. An estimation of more than 700 1-O-acylceramide species, largely isobaric isomers, are present, underscoring the complexity of this Cer family.

SUBMITTER: Lin MH

PROVIDER: S-EPMC5392734 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Linear ion-trap MSn with high-resolution MS reveals structural diversity of 1-O-acylceramide family in mouse epidermis.

Lin Meei-Hua MH Miner Jeffrey H JH Turk John J Hsu Fong-Fu FF

Journal of lipid research 20170202 4

1-O-acylceramide is a new class of epidermal cer-amide (Cer) found in humans and mice. Here, we report an ESI linear ion-trap (LIT) multiple-stage MS (MSn) approach with high resolution toward structural characterization of this lipid family isolated from mice. Molecular species desorbed as the [M + H]+ ions were subjected to LIT MS2 to yield predominately the [M + H - H2O]+ ions, followed by MS3 to cleave the 1-O-acyl residue to ...[more]

PMID: 28154204

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Project description:The discovery of novel protein biomarkers is essential in the clinical setting to enable early disease diagnosis and increase survivability rates. To facilitate differential expression analysis and biomarker discovery, a variety of tandem mass spectrometry (MS/MS)-based protein profiling techniques have been developed. For achieving sensitive detection and accurate quantitation, targeted MS screening approaches, such as multiple reaction monitoring (MRM), have been implemented.MCF-7 breast cancer protein cellular extracts were analyzed by 2D-strong cation exchange (SCX)/reversed phase liquid chromatography (RPLC) separations interfaced to linear ion trap MS detection. MS data were interpreted with the Sequest-based Bioworks software (Thermo Electron). In-house developed Perl-scripts were used to calculate the spectral counts and the representative fragment ions for each peptide.In this work, we report on the generation of a library of 9,677 peptides (p < 0.001), representing approximately 1,572 proteins from human breast cancer cells, that can be used for MRM/MS-based biomarker screening studies. For each protein, the library provides the number and sequence of detectable peptides, the charge state, the spectral count, the molecular weight, the parameters that characterize the quality of the tandem mass spectrum (p-value, DeltaM, Xcorr, DeltaCn, Sp, no. of matching a, b, y ions in the spectrum), the retention time, and the top 10 most intense product ions that correspond to a given peptide. Only proteins identified by at least two spectral counts are listed. The experimental distribution of protein frequencies, as a function of molecular weight, closely matched the theoretical distribution of proteins in the human proteome, as provided in the SwissProt database. The amino acid sequence coverage of the identified proteins ranged from 0.04% to 98.3%. The highest-abundance proteins in the cellular extract had a molecular weight (MW)<50,000.Preliminary experiments have demonstrated that putative biomarkers, that are not detectable by conventional data dependent MS acquisition methods in complex un-fractionated samples, can be reliable identified with the information provided in this library. Based on the spectral count, the quality of a tandem mass spectrum and the m/z values for a parent peptide and its most abundant daughter ions, MRM conditions can be selected to enable the detection of target peptides and proteins.

Dataset Information

Linear ion-trap MSⁿ with high-resolution MS reveals structural diversity of 1-O-acylceramide family in mouse epidermis.

Publications

Linear ion-trap MS<sup>n</sup> with high-resolution MS reveals structural diversity of 1-O-acylceramide family in mouse epidermis.

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