Comparative Analysis of INLIGHT™-labeled Enzymatically Depolymerized Heparin by Reverse-phase Chromatography and High-performance Mass Spectrometry

Mangrum, J.B.; Mehta, A.Y.; Alabbas, A.B., et al.

In an effort to overcome the analytical and technical challenges associated with glycosaminoglycan (GAG) analysis, Mangrum and colleagues applied the INLIGHT™ (individuality normalization when labeling with glycan hydrazide tags) labeling strategy to characterize and quantitatively analyze heparin digests (digHep). Experimentally, the digHep samples were independently tagged with a light (¹²C₆) or heavy (¹³C₆) hydrazide reagent (phenyl 2-GPN) before equimolar mixing and comparative analysis by reversed-phase LC-MS and -MS/MS. In this proof of principle application, the novel extension of the INLIGHT technology from the branched N-glycan analysis to linear sulfated polysaccharides provided sufficient hydrophobicity to show chromatographic resolution up to octamers, while the stable isotope tags provided a means to confidently identify digested heparin oligomers (i.e., matched pair). Given the lack of bioinformatic tools used to identify and quantify sulfated linear polysacchardies, the INLIGHT reagents proved critical to identify heparin oligomers with capacity for relative quantification in biological samples.