| Heparin/heparin sulfates are carbohydrate polyionic polymers that participate in a host of critically important biological processes such as blood anticoagulation, pathogen infection, cell differentiation, growth, migration and inflammation, to mention a few. A century has passed since heparin's initial discovery with a fair understanding of its overall composition. Unfortunately, there has been no structural work at the detailed chemical level that might support a synthetic effort. In this study, I utilize a chemical derivatization strategy (dual permethylation) that imparts isotopic structural specificity, which can be followed by step-wise disassembly in an ion trap mass spectrometer, (MSn). A set of analytical protocols is described that introduces a probable sequencing strategy for heparin analogs. Following derivatization, the O-sulfo groups are converted to deutero methyl esters and the N-sulfo groups are converted to N-deutero acetyl groups. Mass shifts among derivatized products with differential isotopic labeling verified the numbers of N-sulfo groups and O-sulfo groups, while sulfo group positions were characterized by MSn analysis on selected precursor ions. Documentation has been studied with simple heparin/heparin sulfates disaccharides and a synthetic octasulfated pentasaccharide (ArixtraRTM). Determination of this analogous composition, sequence and sulfonation patterns were accomplished by MSn. The protocols provide unique insights into monomer sequence with monomer structural detail observed by cross-ring fragments often resolved by deuterated isotopic labeling. |
