Identification of neutral and acidic glycosphingolipids in the human dermal fibroblasts.

Skin fibroblasts are recognized as a valuable model of primary human cells able of mirroring the chronological and biological aging. Here, a lipidomic study of glycosphingolipids (GSL) occurring in the easily accessible human dermal fibroblasts (HDF) is presented. Reversed-phase liquid chromatography with negative electrospray ionization (RPLC-ESI) coupled to either orbitrap or linear ion-trap multiple-stage mass spectrometry was applied to characterize GSL in commercially adult and neonatal primary human fibroblast cells and in skin samples taken from an adult volunteer. Collision-induced dissociation in negative ion mode allowed us to get information on the monosaccharide number and ceramide composition, whereas tandem mass spectra on the ceramide anion was useful to identify the sphingoid base. Nearly sixty endogenous GSL species were successfully recognized, namely 33 hexosyl-ceramides (i.e., HexCer, Hex2Cer and Hex3Cer) and 24 gangliosides as monosialic acid GM1, GM2 and GM3, along with 5 globosides Gb4. An average content of GSLs was attained and the most representative GSL in skin fibroblasts were Hex3Cer, also known as Gb3Cer, followed by Gb4, HexCer and Hex2Cer , while gangliosides were barely quantifiable. The most abundant GSLs in the examined cell lines share the same ceramide base (i.e. d18:1) and the relative content was d18:1/24:1 > d18:1/24:0 > d18:1/16:0 > d18:1/22:0.

Application of direct HPTLC-MALDI for the qualitative and quantitative profiling of neutral and acidic glycosphingolipids: the case of NEU3 overexpressing C2C12 murine myoblasts.

Glycosphingolipids (GSLs) are a class of ubiquitous lipids characterized by a wide structural repertoire and a variety of functional implications. Importantly, altered levels have been correlated with different diseases, suggesting their crucial role in health. Conventional methods for the characterization and quantification are based on high-performance TLC (HPTLC) separation and comparison with the migration distance of standard samples or on MS. We set up and herein report the application of an ImagePrep method for glycosphingolipids qualitative and quantitative profiling through direct HPTLC-MALDI with particular application to wild-type and NEU3 sialidase-overexpressing C2C12 myoblasts. Lipids were analyzed by HPTLC, coupled with MALDI-TOF, and the resulting GSLs profiles were compared to the [³H]sphingolipids HPTLC patterns obtained after metabolic radiolabeling. GSLs detection by HPTLC-MALDI was optimized by testing different methods for matrix delivery and by performing quantitative analyses using serial dilutions of GSLs standards. Through this approach an accurate analysis of each variant of neutral and acidic GSLs, including the detection of different fatty-acid chain variants for each GSL, was provided and these results demonstrated that HPTLC-MALDI is an easy and high-throughput analytical method for GSLs profiling, suggesting its use for an early detection of markers in different diseases, including cancer and heart ischemia.

Automated normal phase nano high performance liquid chromatography/matrix assisted laser desorption/ionization mass spectrometry for analysis of neutral and acidic glycosphingolipids.

The coupling of nano high-performance liquid chromatography (nanoHPLC) with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) via an automatic spotting roboter was developed and adapted for the first time for the analysis of complex mixtures of glycosphingolipids (GSLs). The 2,5-dihydroxybenzoic acid and 6-azo-2-thiothymine matrix systems were adjusted to concurrently meet the requirements for reproducible and homogeneous crystal formation with the liquid chromatography (LC) eluent under the variable LC solvent composition over the course gradient and high ionization efficiency of the GSL species, without the need for recrystallization. Precise adjustment of the automatic spotting parameters in terms of matrix flow rate, on-tip collection time of the matrix/LC eluent solution and the matrix spotting mode, i.e., continuous and discontinuous, was accomplished to collect individually nanoHPLC-separated species within distinct spots and consequently recover by MALDI MS screening all major and minor GSL species in the mixtures. The nanoHPLC/MALDI MS coupling protocol was developed and applied to a mixture of neutral GSLs purified from human erythrocytes and a monosialoganglioside mixture expressed by the murine MDAY-D2 cell line. Additionally, on-line nanoHPLC/MALDI doping with lithium cations of individually separated neutral GSLs was introduced to enhance data interpretation of the GSL MS pattern, while preserving the same level of information and ultimately to enhance structural assignment of components of interest. The method is demonstrated to be highly sensitive, reaching the low femtomole level of detection of individual GSL species and is highlighted as a versatile analytical tool for glycolipidomic studies. [figure: see text]

Identification of GM3 as a marker of therapy-resistant periradicular lesions.

The purpose of this study was to analyze the profile of glycosphingolipids (GSLs) in periradicular lesions refractory to endodontic treatment. Sixteen periapical lesions were removed surgically from patients (experimental group) and compared with 10 samples of periodontal ligament removed from extracted intact third molars (control group). After the GSLs extraction and purification procedures were performed the neutral and acidic GSL fractions were analyzed by high-performance thin-layer chromatography and quantified by densitometry. Data reported herein show that: (i) tissues in the experimental group presented about twice as much GSLs as the control group; (ii) lesion tissues express lactoneotetraosylceramide, and lactofucopentaosyl (IV) ceramide, whereas these neutral GSLs are absent in normal tissues; and (iii) normal tissues express GT1b, whereas lesions cells do not express this ganglioside. In contrast lesion tissues express GM3, which is conspicuously absent in normal tissues.