Tandem MS of heparin oligosaccharides
The heparin compound class represents a particular challenge owing to the high degree of sulfation. It has the highest number of sulfate groups (2-3 on average) per disaccharide unit among the glycosaminoglycan (GAG) classes. Low energy CID remains the approach for biomolecular dissociation that is most amenable to generation of high throughput data. For GAG oligosaccharides, it is desirable to produce the highest (absolute value) negative ion charge states, so as to stabilize sulfate groups during CID. This approach has proven effective for chondroitin sulfate and heparan sulfate, both of which classes have fewer sulfate groups per disaccharide unit than the heparins. For heparin oligosaccharides, it is generally not possible to produce sufficiently high charge states using on-line LC/MS to enable production of abundant backbone dissociation product ions. Charge states are considerably higher when using static nano-ESI, due to the decreased droplet size. Heparin was partially depolymerized using heparin lyase I, and the products fractionated using preparative size exclusion chromatography. The oligosaccharides were analyzed by tandem MS using static nano-ESI using an Advion robot and the LTQ-Orbitrap. The results showed that glycosidic bond dissociation was the major product ion pathway for ions in which the (sulfate number)/(charge state) ratio (S/z) was between 1 and 2. Losses of sulfate increased above S/z 1.7. It was not possible to generate ions with S/z less than 1 for the heparin class. For heparan sulfate oligosaccharides, ions ranging from S/z 0.5 to 1.5 were generated. The data showed that losses of sulfate increased above S/z 1.2. We believe that the increased density of sulfate groups on heparin oligosaccharides results in charge-charge repulsion that strain glycosidic bonds, allowing effective backbone dissociation at slightly higher S/z values than for the heparan sulfate class. These results demonstrate parameters for acquisition of off-line tandem mass spectra for the heparin GAG class.
