Top–down glycolipidomics: fragmentation analysis of ganglioside oligosaccharide core and ceramide moiety by chip‐nanoelectrospray collision‐induced dissociation MS 2 –MS 6

We developed a straightforward approach for high‐throughput top–down glycolipidomics based on fully automated chip‐nanoelectrospray (nanoESI) high‐capacity ion trap (HCT) multistage mass spectrometry (MS n ) by collision‐induced dissociation (CID) in the negative ion mode. The method was optimized and tested on a polysialylated ganglioside fraction (GT1b), which was profiled by MS 1 and sequenced in tandem MS up to MS 6 in the same experiment. Screening of the fraction in the MS 1 mode indicated the occurrence of six [M − 2H] 2− ions which, according to calculation, support 13 GT1 variants differing in their relative molecular mass due to dissimilar ceramide (Cer) constitutions. By stepwise CID MS 2 –MS 5 on the doubly charged ion at m / z 1077.20 corresponding to a ubiquitous GT1b structure, the complete characterization of its oligosaccharide core including the identification of sialylation sites was achieved. Structure of the lipid moiety was further elucidated by CID MS 6 analysis carried out using the Y 0 fragment ion, detected in MS 5 , as a precursor. MS 6 fragmentation resulted in a pattern supporting a single ceramide form having the less common (d20 : 1/18 : 0) configuration. The entire top–down experiment was performed in a high‐throughput regime in less than 3 min of measurement, with an analysis sensitivity situated in the subpicomolar range. Copyright © 2009 John Wiley & Sons, Ltd.