Abstract
Structural characterization of the microheterogeneity of heparin, heparan sulfate, and other glycosaminoglycans is a major analytical challenge. We present the use of a stable isotope-labeled hydrazide tag (INLIGHT (TM)) with high-resolution/accurate mass (HRAM) reverse-phase LC-MS/MS, which was recently introduced for detailed study of N-glycan heterogeneity, to characterize heparinase-digested heparin (digHep) products without the use of semi-volatile ion pairing reagents. Using both full scan LC-MS and data-dependent LC-MS/MS, we identified 116 unique digHep species, a feat possible because of INLIGHT (TM) labeling. Of these, 83 digHep products were structurally identified, including the 12 standard disaccharides as well as 34 tetra- (DP4), 26 hexa- (DP6), 21 octa- (DP8), and 2 decasaccharides (DP10). Each of the 116 digHep species co-eluted with both light and heavy INLIGHT (TM) tags (L/H-avg = 1.039 +/- 0.163); thus enhancing confidence in their identification via MS and MS/MS. This work sets the foundation for INLIGHT (TM)-based comparative analyses of different forms of heparin, heparan sulfate, and other GAGs with high quantitative precision using mainstay reverse-phase HRAM LC-MS/MS.