TY - JOUR
T1 - High-performance liquid chromatography-mass spectrometry for mapping and sequencing glycosaminoglycan-derived oligosaccharides
AU - Volpi, Nicola
AU - Linhardt, Robert J.
N1 - Funding Information:
acKnowleDGMents This work was supported in part through grants from the US National Institutes of Health (GM38060, HL096972, HL101721 and GM090127) to R.J.L.
PY - 2010/6
Y1 - 2010/6
N2 - Glycosaminoglycans (GAGs) have proven to be very difficult to analyze and characterize because of their high negative charge density, polydispersity and sequence heterogeneity. As the specificity of the interactions between GAGs and proteins results from the structure of these polysaccharides, an understanding of GAG structure is essential for developing a structure-activity relationship. Electrospray ionization (ESI) mass spectrometry (MS) is particularly promising for the analysis of oligosaccharides chemically or enzymatically generated by GAGs because of its relatively soft ionization capacity. Furthermore, on-line high-performance liquid chromatography (HPLC)-MS greatly enhances the characterization of complex mixtures of GAG-derived oligosaccharides, providing important structural information and affording their disaccharide composition. A detailed protocol for producing oligosaccharides from various GAGs, using controlled, specific enzymatic or chemical depolymerization, is presented, together with their HPLC separation, using volatile reversed-phase ion-pairing reagents and on-line ESI-MS structural identification. This analysis provides an oligosaccharide map together with sequence information from a reading frame beginning at the nonreducing end of the GAG chains. The preparation of oligosaccharides can be carried out in 10 h, with subsequent HPLC analysis in 1-2 h and HPLC-MS analysis taking another 2 h.
AB - Glycosaminoglycans (GAGs) have proven to be very difficult to analyze and characterize because of their high negative charge density, polydispersity and sequence heterogeneity. As the specificity of the interactions between GAGs and proteins results from the structure of these polysaccharides, an understanding of GAG structure is essential for developing a structure-activity relationship. Electrospray ionization (ESI) mass spectrometry (MS) is particularly promising for the analysis of oligosaccharides chemically or enzymatically generated by GAGs because of its relatively soft ionization capacity. Furthermore, on-line high-performance liquid chromatography (HPLC)-MS greatly enhances the characterization of complex mixtures of GAG-derived oligosaccharides, providing important structural information and affording their disaccharide composition. A detailed protocol for producing oligosaccharides from various GAGs, using controlled, specific enzymatic or chemical depolymerization, is presented, together with their HPLC separation, using volatile reversed-phase ion-pairing reagents and on-line ESI-MS structural identification. This analysis provides an oligosaccharide map together with sequence information from a reading frame beginning at the nonreducing end of the GAG chains. The preparation of oligosaccharides can be carried out in 10 h, with subsequent HPLC analysis in 1-2 h and HPLC-MS analysis taking another 2 h.
UR - https://www.scopus.com/pages/publications/77952149942
U2 - 10.1038/nprot.2010.48
DO - 10.1038/nprot.2010.48
M3 - Article
C2 - 20448545
AN - SCOPUS:77952149942
SN - 1754-2189
VL - 5
SP - 993
EP - 1004
JO - Nature Protocols
JF - Nature Protocols
IS - 6
ER -