title: Comparative analysis of INLIGHT™-labeled enzymatically depolymerized heparin by reverse-phase chromatography and high-performance mass spectrometry.
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™) 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™ 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™ tags (L/Havg= 1.039 ± 0.163); thus enhancing confidence in their identification via MS and MS/MS. This work sets the foundation for INLIGHT™-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. Graphical Abstract Reducing end labeling strategy for mapping depolymerized heparin/heparan sulfate products by reverse-phase LC-MS/MS.
authors.affiliation: Food and Drug Administration, College Park, MD, 20740-3835, USA.
authors.forename: John B
authors.initials: JB
authors.lastname: Mangrum
authors.affiliation: Department of Medicinal Chemistry, Virginia Commonwealth University (VCU) School of Pharmacy, 800 E. Leigh Street, P.O. Box 980540, Richmond, VA, 23298-0540, USA.
authors.forename: Akul Y
authors.initials: AY
authors.lastname: Mehta
authors.affiliation: Department of Medicinal Chemistry, Virginia Commonwealth University (VCU) School of Pharmacy, 800 E. Leigh Street, P.O. Box 980540, Richmond, VA, 23298-0540, USA.
authors.forename: Alhumaidi B
authors.initials: AB
authors.lastname: Alabbas
authors.affiliation: Department of Medicinal Chemistry, Virginia Commonwealth University (VCU) School of Pharmacy, 800 E. Leigh Street, P.O. Box 980540, Richmond, VA, 23298-0540, USA.|Institute for Structural Biology, Drug Discovery, and Development, Virginia Commonwealth University (VCU) School of Pharmacy, 800 E. Leigh Street, Suite 212, Richmond, VA, 23219, USA.
authors.forename: Umesh R
authors.initials: UR
authors.lastname: Desai
authors.affiliation: Institute for Structural Biology, Drug Discovery, and Development, Virginia Commonwealth University (VCU) School of Pharmacy, 800 E. Leigh Street, Suite 212, Richmond, VA, 23219, USA. amhawkridge@vcu.edu.|Department of Pharmaceutics, Virginia Commonwealth University (VCU) School of Pharmacy, 410 North 12th Street, P.O. Box 980533, Richmond, VA, 23298-0533, USA. amhawkridge@vcu.edu.|Department of Pharmacotherapy and Outcomes Science, Virginia Commonwealth University (VCU) School of Pharmacy, 410 North 12th Street, P.O. Box 980533, Richmond, VA, 23298-0533, USA. amhawkridge@vcu.edu.
authors.forename: Adam M
authors.initials: AM
authors.lastname: Hawkridge
mesh_terms: D056148:Chromatography, Reverse-Phase; D006025:Glycosaminoglycans / Q000737:chemistry; D006493:Heparin / Q000032:analysis / Q000737:chemistry*; D019763:Heparin Lyase / Q000737:chemistry; D006497:Heparan Sulfate / Q000737:chemistry; D058105:Polymerization; D053719:Tandem Mass Spectrometry
chemical_list: D006025:Glycosaminoglycans; D006493:Heparin; D019763:Heparin Lyase; D006497:Heparan Sulfate
keywords: Chemical derivatization; Glycosaminoglycan; Heparin; Mass spectrometry/ICP-MS