Analysis of heparan sulfate oligosaccharides by nano-electrospray ionization mass spectrometry

A highly sensitive method to identify and quantify heparan sulfate (HS) oli gosaccharides by using nano-electrospray ionization mass spectrometry (nESI -MS) is described. The new approach allows us to detect approximately 50 nM of a chemically synthesized pentasaccharide with a structure of GlcNS6S-Gl cA-GlcNS6S-IdoA2-S-GlcNS6SOMe (3-OH pentasaccharide), Typically, solutions were infused for a total of 5 min, at an average flow rate of 30 nl/min, an d the remaining sample was recovered from the nanovial, The spectra shown w ere obtained by summing scans for 1-3 min. Hence, our data indicated that a s little as 3 x 10(-15) mole of the pentasaccharide was consumed to obtain a reasonable spectrum at the concentration as low as 50 nM. In addition, we found a linear relationship between the relative response of the molecular ion and the concentration of the analyzed 3-OH pentasaccharide, demonstrat ing that this approach can be used to determine the amount of HS oligosacch arides, To this end, a 3-O-sulfated pentasaccharide was prepared by incubat ing the 3-OH pentasaccharide with purified HS 3-O-sulfotransferase-1 and 3' -phosphoadenosine-5'-phospho[S-35]sulfate. The resulting 3-O-sulfated penta saccharide was purified and analyzed by nESI-MS. Based on the standard curv e constructed with the 3-OH pentasaccharide, we calculated the concentratio n of the 3-O-sulfated pentasaccharide by the relative response. The result indicates that this value is very close to the value measured by [S-35]sulf ate radioactivity. In conclusion, nESI-MS provides both high sensitivity an d the capacity to quantify HSs, This approach is likely to become a very im portant tool for structural analysis and sequencing of HS and heparin oligo saccharides.