Automated and Rapid Characterization of Low-Molecular-Weight Heparin Oligosaccharides using SimGlycan software v. 5.90

February 15, 2018

Unfractionated Heparin (UH) is an anticoagulant formulated of anionic, sulfated glycosaminoglycan. However, during infusion, this naturally occurring anticoagulant has been associated with various health problems. In contrast, low molecular weight heparins (LMWH) such as Enoxaparin, Parnaparin, Certoparin, Deltaparin, etc., are relatively efficient and safer. Therefore, an increasing demand to study these LMWH as new regulatory drugs has been on the rise. However, due to structural complexity, characterization of these LMWH remained a distant dream. Though liquid chromatography based mass-spectrometry can be a sensitive technique to study these oligosaccharides, lack of adequate software tools to study the humongous and complex data generated by LC/MS makes it a major challenge.

In an attempt to rapidly identify LWMH glycans accurately, a database containing LMWH glycans curated from the published literature was created in SimGlycan v.5.90. We successfully constructed a database consisting of 317 probable structural isomers of LMWH glycans with all the reported core motifs. Each of the core motifs were substituted with SO3, Ac, H to take into account of the heterogeneity of the probable structures. The custom database is used as an MS/MS search template in order to ensure correct assignment of glycans.

The digested mixture was prepared by mixing 20μL of Enoxaparin sodium (20mg/ml in deionized water) (Zhang et. al., (2013), followed by filtering using 0.45mm filter. The mixture was used as final test solution for RPIP-UPLC-MS analysis. For LC analysis, Shimadzu HPLC System on Agilent SB C18 (4.6 x 100 mm, and 1.8um columns connected in serial) was used. The mobile phase compositions were: A (Water with 15mMHexylamine) and B (75% ACN with 15mM Hexylamine). Data acquisition was performed with a TripleTOF 5600 (SCIEX, Concord, ON) in negative electro-spray ionization mode.

The LC-MS/MS data was subjected to SimGlycan database search for automated annotation of glycan structures using 25 ppm tolerance for both the precursor and product ions. SimGlycan v.5.90 showed a good coverage by annotating 84 Enoxaparin oligosaccharides. The presence of modifications in carbohydrate residues such as acetylation, different sulphation degrees were also captured using SimGlycan generated MS/MS spectra annotated with fragment ions (fragment ion nomenclature: cartoons; successive loss of carbohydrate residues; Domon and Costello) of the identified glycans. The results consisting the lists of the annotated glycans can be exported to MS Excel.

For more details on experimental methods, and detailed structures, please refer to the poster titled "Automated and Rapid Characterization of Low-Molecular-Weight Heparin Oligosaccharides Using TripleTOF 5600 Mass Spectrometer with SimGlycan Software".

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