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.