Database and data analysis application for structural characterization of gangliosides and sulfated glycosphingolipids by negative ion mass spectrometry

Abstract

Gangliosides and sulfated glycosphingolipids, as building and functional components of animal cell mem-branes, participate in cell-to-cell interactions and signaling, but also in changes of cell architecture due to different pathophysiological events. In order to enable higher throughput and to facilitate structural characterization of gangliosides/sulfo-glycosphingolipids (GSL) and their neutral GSL counterparts by negative ion mass spectrometry (MS) and tandem MS techniques, a database and data analysis applica-tion have been developed. In silico developed glycosphingolipid database considers a high diversity of ceramide compositions, several sialic acid types (N-acetylneuraminic acid, N-glycolylneuraminic acid and 2-keto-3-deoxynononic acid) as well as possible additional substitutions/modifications of glycosphingolipids, such as O-acetylation, de-N-acetylation, fucosylation, glucuronosylation, sulfation, attachment of repeating terminal hexose-N-acetylhexosamine-(Hex-HexNAc-) 1-6 extension, and possi-ble lactone forms. Data analysis application, named GSL-finder, enables correlation of negative ion MS and/or low-energy tandem MS spectra with the database structures. The GSLdatabase construction and the GSL-finder application searching rules are explained. Validation conducted on GD1a fraction as well as on complex mixtures of native gangliosides isolated from different mammalian brain tissues (human fetal and adult brain, and calf brain tissue) demonstrated agreement with previous studies. Plain, fast, and automated routine for structural characterization of gangliosides/sulfated glycosphingolipids and their neutral GSL counterparts described here could facilitate and improve mass spectrometric analysis of complex glycosphingolipid mixtures originating from variety of normal and pathological biomaterial, where it is known that distinctive changes in glycosphingolipid composition occur.

Characterization and Modeling of the Collision Induced Dissociation Patterns of Deprotonated Glycosphingolipids: Cleavage of the Glycosidic Bond

“…Negative ion mode MS and tandem MS analysis of the GSL samples were performed on the Bruker amaZon ETD ion trap system (Bruker Daltonik GmbH, Bremen, Germany) using the experimental procedure described in the previous publications [10,16]. GSL ion identification was accomplished using the software application BGSL finder^and its accompanying GSL database [16]. Additionally, spectra were manually validated.…”

“…Owing to the nature of extraction and purification procedures used [consult references 10,16] only few of the neutral GSL species (species with unsubstituted glycosyl moiety) were observed (e.g., Supporting Information, S- Figure 1b). However, let us first consider possible pathways of the glycosidic bond cleavage of the neutral GSLs.…”

“…In our previous work we have demonstrated the computationally generated glycosphingolipid (GSL) ion database for negative ion MS and MSMS ion spectra search [16]. The database and accompanying application (GSL-finder) have been successfully applied to the analysis of mice brain gangliosides (sialic acid (Neu5Ac) containing glycosphingolipids) with altered ganglioside biosynthesis, on a commercially available ganglioside fraction, and on complex native ganglioside mixtures isolated from human (fetal and adult) and calf brain tissues [10,16].…”

“…The database and accompanying application (GSL-finder) have been successfully applied to the analysis of mice brain gangliosides (sialic acid (Neu5Ac) containing glycosphingolipids) with altered ganglioside biosynthesis, on a commercially available ganglioside fraction, and on complex native ganglioside mixtures isolated from human (fetal and adult) and calf brain tissues [10,16]. The GSL-finder application and other software tools for mass spectrometric data analysis of glycoconjugates are designed to assign molecular structures to tandem MS spectra [16][17][18]. For this strategy to be effective, fragmentation models utilized in software applications need to implement latest understandings of dissociation mechanisms of GSLs.…”

“…A small data set of 75 tandem MS spectra collected from previous studies [10,16] was used to suggest fragmentation behavior of GSLs. The proposed fragmentation pathways have been computationally assessed on a GSL level.…”