Napredna pretraga

Pregled bibliografske jedinice broj: 409543

Compositional and structural analysis of gangliosides in human cerebrospinal fluid by chip-based nanoelectrospray ionization tandem mass spectrometry


Serb, Alina; Schiopu, Catalin; Marinčić, Dragana; Vukelić, Željka; Zamfir, Alina D.
Compositional and structural analysis of gangliosides in human cerebrospinal fluid by chip-based nanoelectrospray ionization tandem mass spectrometry // 57th ASMS Conference on Mass Spectrometry : Book of Abstracts
Philadelphia, 2009. (poster, međunarodna recenzija, sažetak, znanstveni)


Naslov
Compositional and structural analysis of gangliosides in human cerebrospinal fluid by chip-based nanoelectrospray ionization tandem mass spectrometry

Autori
Serb, Alina ; Schiopu, Catalin ; Marinčić, Dragana ; Vukelić, Željka ; Zamfir, Alina D.

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
57th ASMS Conference on Mass Spectrometry : Book of Abstracts / - Philadelphia, 2009

Skup
ASMS Conference on Mass Spectrometry (57 ; 2009)

Mjesto i datum
Philadelphia, Pennsylvania, SAD, 31.05.-04.06.2009

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
Gangliosides; human cerebrospinal fluid; nanoESI HCT MS

Sažetak
Sialylated glycosphingolipids, gangliosides, are primarily building plasma membrane components of all mammalian cell types ; the highest concentrations are present in the brain cells. Ganglioside expression has a cellular specificity during the development process and in close connection with the differentiation of cells. Brain gangliosides shed into the extracellular space and thus are found in the cerebrospinal fluid (CSF). Determination of ganglioside expression and structure in CSF is of high biological and clinical importance since alterations in the CSF concentrations of gangliosides was previously reported as markers of degenerative processes affecting neuronal membranes. Here, we report on the first application of mass spectrometry (MS) for compositional and structural analysis of native gangliosides from human cerebrospinal fluid. Gangliosides were extracted from 5 mL of normal human CSF. After CSF volume reduction by ultrafiltration, gangliosides were extracted with methanol/chloroform, separated and quantified by high performance thin layer chromatography and direct densitometry. For MS analysis purified ganglioside mixture was dissolved in methanol to a concentration of about 5 pmol/μ L. Fully automated chip-based nanoelectrospray (nanoESI) was performed on a NanoMate robot (Advion BioSciences, Ithaca, USA) coupled to a high capacity ion trap (HCT) MS. NanoMate HCT MS system was tuned to operate in the negative ion mode. Electrospray was initiated by applying a voltage of -0.90 kV on the pipette tip, and 1.10 psi nitrogen back pressure. MS2-MS5 was performed by collision-induced dissociation (CID) using He as collision gas. 5 μ L of working sample solution were loaded onto the NanoMate 96-microtiter plate. The robot was programmed to aspirate the whole volume of sample, followed by 2 µ ; L of air into the pipette tip and deliver the sample to the inlet side of the 400 microchip. All mass spectra were acquired in the m/z range 100-3000, with a scan speed of 8000 m/z per second. Screening mass spectrum obtained under these conditions enabled the detection of more than 50 individual species and revealed that CSF ganglioside pattern resembles that of brain gangliosides, particularly cerebellum gangliosides. Almost all oligosaccharide portion-defined ganglioside forms were represented by several species differing in the composition of their ceramide residues. The major found structural forms were GM3, GM2, GD3, GM1/nLM1/LM1, GD1/nLD1 and GT1. GD2 and GT3 species were present with lower abundance. Polysialylated structures, GQ1 and GP1, fucosylated, O-GalNAc-modified as well as alkali-labile O-acetylated and lactonized ganglioside species were found with similar relative abundance as previously observed in the crude ganglioside mixture from adult human cerebellum analyzed under identical conditions. In particular, biologically relevant O-acetylated and fucosylated GD1, GT1 and GQ1 exhibiting high heterogeneity in their ceramide motifs were evidenced. Fucose-modified GD1 was detected in the highest number (five) of glycoforms. According to MS screening results, under the employed conditions a high sensitivity ganglioside mixture analysis without compromising the ionization of minor, biologically relevant components, could be achieved by nanoESI-chip MS approach. Furthermore, CID MS2-MS5 sequencing patterns of selected precursor ions corresponding to polysialylated, Fuc- and O-Ac modified species yielded a complete set of structural data within short analysis time and with drastically reduced sample consumption. MS2-MS5 allowed a detailed structural characterization of the oligosaccharide core and ceramide moiety as well as collecting specific data upon sialic acid localization leading to isomer identification. In this study we report on the first mass spectrometric investigation of ganglioside composition and structure in human cerebrospinal fluid.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti



POVEZANOST RADA


Projekt / tema
108-1081870-2415 - Strukturno-funkcionalna glikolipidomika moždanog razvitka i maligne alteracije (Željka Vukelić, )

Ustanove
Medicinski fakultet, Zagreb