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

Abstract

A highly sensitive method to identify and quantify heparan sulfate (HS) oligosaccharides 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-GlcA-GlcNS6S-IdoA2S-GlcNS6SOMe (3-OH pentasaccharide). Typically, solutions were infused for a total of 5 min, at an average flow rate of 30 nl/min, and the remaining sample was recovered from the nanovial. The spectra shown were obtained by summing scans for 1--3 min. Hence, our data indicated that as 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, demonstrating that this approach can be used to determine the amount of HS oligosaccharides. To this end, a 3-O-sulfated pentasaccharide was prepared by incubating the 3-OH pentasaccharide with purified HS 3-O-sulfotransferase-1 and 3'-phosphoadenosine-5'-phospho[(35)S]sulfate. The resulting 3-O-sulfated pentasaccharide was purified and analyzed by nESI-MS. Based on the standard curve constructed with the 3-OH pentasaccharide, we calculated the concentration of the 3-O-sulfated pentasaccharide by the relative response. The result indicates that this value is very close to the value measured by [(35)S]sulfate radioactivity. In conclusion, nESI-MS provides both high sensitivity and the capacity to quantify HSs. This approach is likely to become a very important tool for structural analysis and sequencing of HS and heparin oligosaccharides.

Characterization of a Heparan Sulfate Octasaccharide That Binds to Herpes Simplex Virus Type 1 Glycoprotein D

“…4A). We confirmed that the signals at m/z 648.8 and 486.4 contain sulfate groups by using neutral loss experiments as described in a prior publication (29). Briefly, the isolated ions were sequentially admitted to a collision cell filled with argon under controlled energy conditions, resulting in limited dissociation.…”

“…The MALDI-MS spectra were acquired in the linear mode using a Perseptive Biosystems Voyager Elite reflectron time-of-flight instrument fitted with a 337-nm laser as described elsewhere (28). The nESI-MS analysis was carried out using a Micromass Quattro II with QhQ geometry, a Z-spray source, and pulled borosilicate glass nanovials (29). In the neutral loss scan, MS/MS spectra were obtained by scanning Q1 and Q3 with an offset of 26.7 or 20 m/z in their scan cycles, corresponding to the loss of sulfate from the triple or quadruple charged octasaccharide, respectively (29).…”

“…The nESI-MS analysis was carried out using a Micromass Quattro II with QhQ geometry, a Z-spray source, and pulled borosilicate glass nanovials (29). In the neutral loss scan, MS/MS spectra were obtained by scanning Q1 and Q3 with an offset of 26.7 or 20 m/z in their scan cycles, corresponding to the loss of sulfate from the triple or quadruple charged octasaccharide, respectively (29). To obtain a high quality nESI-MS spectrum, the purified octasaccharide was further dialyzed against 25 mM ammonium acetate (purity of ammonium acetate is 99.9999%, Aldrich) using MWCO 13,000 hollow fiber dialysis tubing (Spectrum).…”

“…The pentasaccharide was prepared by incubating acetic anhydride with a pentasaccharide with a structure of GlcNH 2 -GlcUA-[3-35 S]GlcNS3S6S-IdoUA2S-GlcNS6SOMe. The latter pentasaccharide was generated from GlcNS6S-GlcUA-[3-35 S]GlcNS3S6S-IdoUA2S-GlcNS6SOMe (29) by sequential digestions by glucosamine-6-sulfatase and sulfamidase. 8 The tetrasaccharide was prepared by incubating purified fraction D with hydrazine for deacetylation.…”

Chemoenzymatic Design of Heparan Sulfate Oligosaccharides*

“…(GlcUA-GlcNS3S6S) 5 -GlcUA-AnMan 145 Ϯ 24 10 (GlcUA-GlcNS6S) 5 -GlcUA-AnMan Ͼ100,000 Arixtra a GlcNS6S-GlcUA-GlcNS3S6S-IdoUA2S-GlcNS6S-OMe 33 Recomparin b Polysaccharide, no defined structure 57 a The 35 S-labeled Arixtra was prepared as described in a paper published previously (33). b The binding affinity of Recomparin to AT was taken from our previous publication (6).…”