Detailed Characterization of Carbohydrate Linkage and Sequence in an Ion Trap Mass Spectrometer: Glycosphingolipids

Electrospray ionization with a quadrupole ion trap (qIT) mass analyzer has been utilized to ascertain structural detail obtained with glycoconjugate samples. In this report, an isomeric disialyl glycosphingolipid sample extracted from human brain tissue was evaluated for sequence, branching, and linkage information. Results were obtained that were qualitatively comparable with triple-quadrupole instruments (Q1q2Q3) with major carbohydrate fragments from C1-O glycosidic rupture and additional fragments that provided a determination of sphingosine andN-acyl heterogeneity of the ceramide moiety. In unique contrast, however, the qIT extended carbohydrate understanding through multistep mass spectrometric (MSn) studies providing for the first time pyran cross-ring cleavages that define the interresidue linkage structure for glycolipids. This was achieved by selecting secondary fragments (MS2) free from the energy sinks of facile bond rupture that dominate product ion spectra. Isolation and activation of these substructures result in a more uniform distribution of fragments, providing structural insights previously inaccessible by tandem mass spectrometry.