TY - JOUR
T1 - Separation of biosynthetic oligosaccharide branch isomers using high- performance liquid chromatography on a porous two-dimensional graphite stationary phase
AU - Lipniunas, Peter H.
AU - Neville, David C.A.
AU - Trimble, Robert B.
AU - Townsend, R. Reid
N1 - Funding Information:
This work was supported by the National Institutes of Health (RR01614 and GM23900) and the Wenner±Gren Foundation (to P.L.). The mass spectra were obtained at the UCSF Mass Spectrometry Facility supported by the Biomedical Research Technology Program of the National Center for Research Resources (NIH NCRR BRTP RR01614 and RR08282). The VG TofSpec SE was partially supported by Micromass (Beverly, MA).
PY - 1996/12/15
Y1 - 1996/12/15
N2 - Oligomannosidic branch isomers (structures differing only in the branch location of a single residue) which are biosynthetic intermediates in yeast and higher eukaryotics have been separated using high-performance liquid chromatography (HPLC) on porous graphatized carbon (PGC) columns, a stationary phase of two-dimensional crystalline carbon. A mixture of two Man6GlcNAc isomers from IgM, which was determined from 1H NMR analysis, was completely separated by PGC-HPLC. Mixtures of larger yeast oligomannosidic branch isomers were also chromatographically resolved using PGC-HPLC. Man10GlcNAc and Man11GlcNAc species from invertase expressed in Pichia pastoris showed three and five peak fractions, respectively, by PGC-HPLC in agreement with the number of isomeric forms from one- and two-dimensional 1H NMR analyses of the individual sized fractions (Trimble, R.B., Atkinson, P.H., Tschopp, J.R., Townsend, R.R., and Maley, F. (1991) J. Biol. Chem. 266, 22807-22817). Selected peak fractions were further analyzed to confirm assignments using matrix-assisted laser-desorption mass spectrometry after digestion with an α(1 → 2)-specific mannosidase (Aspergillus saitoi). PGC- HPLC should prove invaluable for the preparation of singular oligosaccharides to define exoglycosidase and glycosyl transferase branch specificity and for preparing standards to develop more sensitive methods for structural elucidation of oligosaccharides.
AB - Oligomannosidic branch isomers (structures differing only in the branch location of a single residue) which are biosynthetic intermediates in yeast and higher eukaryotics have been separated using high-performance liquid chromatography (HPLC) on porous graphatized carbon (PGC) columns, a stationary phase of two-dimensional crystalline carbon. A mixture of two Man6GlcNAc isomers from IgM, which was determined from 1H NMR analysis, was completely separated by PGC-HPLC. Mixtures of larger yeast oligomannosidic branch isomers were also chromatographically resolved using PGC-HPLC. Man10GlcNAc and Man11GlcNAc species from invertase expressed in Pichia pastoris showed three and five peak fractions, respectively, by PGC-HPLC in agreement with the number of isomeric forms from one- and two-dimensional 1H NMR analyses of the individual sized fractions (Trimble, R.B., Atkinson, P.H., Tschopp, J.R., Townsend, R.R., and Maley, F. (1991) J. Biol. Chem. 266, 22807-22817). Selected peak fractions were further analyzed to confirm assignments using matrix-assisted laser-desorption mass spectrometry after digestion with an α(1 → 2)-specific mannosidase (Aspergillus saitoi). PGC- HPLC should prove invaluable for the preparation of singular oligosaccharides to define exoglycosidase and glycosyl transferase branch specificity and for preparing standards to develop more sensitive methods for structural elucidation of oligosaccharides.
UR - http://www.scopus.com/inward/record.url?scp=0030589719&partnerID=8YFLogxK
U2 - 10.1006/abio.1996.0507
DO - 10.1006/abio.1996.0507
M3 - Article
C2 - 8954551
AN - SCOPUS:0030589719
SN - 0003-2697
VL - 243
SP - 203
EP - 209
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -