FR AB - Science Reference

P85-S
Rapid oligosaccharide mapping using fluorescent
anthranilic acid detection.
S.T. Dhume, S.A. Batz; SmithKline Beecham, 709 Swedeland Rd,
Mailcode UW2960, King of Prussia, PA 19406
Oligosaccharide mapping and characterization methods based on fluorescent
Anthranilic acid (AA, 2-aminobenzoic acid) labeling affords high resolution
and high sensitivity detection of glycans (1). The AA tagging method offers
a significant improvement over other methods and is being rapidly adopted
in the area of glycoprotein analysis.
The oligosaccharide mapping method starting with an N-linked glycoprotein
requires an overnight enzymatic digestion, 1 hour reaction with AA, a purification
step followed by a 2 hour chromatographic run. It is the intent of this
work to allow rapid detection of oligosaccharides while retaining the quality
and reproducibility of the original method.
The enzymatic release of oligosaccharides with the amounts of substrate and
enzyme used is essentially complete in 3 hours. The glycan profiles obtained
with PNGase F incubations between 30 min and 72 hours are similar except
for the lower peak intensities at shorter (�2.5 hours) incubation times.
The purification step to remove excess AA may be omitted. The mapping is
directly applicable to fetuin, a highly sialylated glycoprotein but needed gradient
changes for neutral glycan species to retain resolution, tailing and
quality of maps as those from the original method.
Oligosaccharide mapping was carried out on a short (2.1 mm � 15 cm) polymeric
amine-bonded column allowing reduction in the time of analysis to
about an hour. Initial results with reverse phase chromatography are promising
and also allow 1 hour runs per sample.
The modifications may be used alone or in any combination. Without consideration
of the time for enzyme incubation, the total time saved is more
than 50%. In addition, 80% of HPLC solvent consumption is avoided. The
reproducibility and universal applicability of this method for other oligosaccharides
will be discussed in detail.
1. Anumula, K. R. and Dhume, S. T. (1998) Glycobiology, 8, 685–694.
P87-T
FMN is covalently attached to a specified threonine residue
via phosphate group in the NqrB and NqrC subunits of
Na�-translocating NADH-quinone reductase from
Vibrio alginolyticus.
M. Maeda1, M. Hayashi2, Y. Nakayama2, M. Yasui2, K. Furuishi3, T. Unemoto2; 1Applied Biosystems, Framingham, MA, 2Chiba Univ.,
3Applied Biosystems, 4-5-4 Hatchobori, Chuo-ku, Tokyo 104-0032, Japan
Na �-translocating NADH-quinone reductase (NQR) from Vibrio alginolyticus
is composed of six subunits (NqrA to NqrF). We previously demonstrated that
both NqrB and NqrC subunits contain a flavin cofactor covalently attached
to a threonine residue. Fluorescent peptide fragments derived from the NqrB
and NqrC subunits were applied to a matrix-assisted laser desorption time of
flight (MALDI-TOF) mass spectrometer and covalently attached flavin was
identified to be FMN in both subunits. From post-source decay (PSD) fragmentation
analysis, it was concluded that FMN is attached via phosphate
group to Thr-235 in the NqrB and to Thr-223 in the NqrC subunits. The ester
binding of FMN to the threonine residue reported here is a new type of flavin
attachment to polypeptide.
POSTER ABSTRACTS
ABRF 2001 ABSTRACTS
P86-M
GlycoSuiteDB—a database of glycan structures.
C.A. Cooper, M.J. Harrison, M.R. Wilkins, N.H. Packer; Proteome Systems
Ltd., North Ryde, Australia, 1/35-41 Waterloo Rd, North Ryde, NSW 2113,
Australia
GlycoSuiteDB is a relational database that contains information from the scientific
literature on glycoprotein derived glycan structures, their biological
sources, the literature references used to obtain the information, and the
methods used to determine each glycan structure. The main aims in the construction
of GlycoSuiteDB are to present a consistent, up-to-date and reliable
source of information. The database provides an essential resource for the
glycobiologist and the protein chemist.
GlycoSuiteDB is available on the web at http://www.glycosuite.com. The
web site allows the user to search the database using a combination of composition,
monoisotopic or average mass, protein name, SWISS-PROT/TrEMBL
accession number, species, biological system, tissue or cell type. There are
at present no restrictions on the use of GlycoSuiteDB by non-profit organisations
as long as its content is not modified in any way. Usage by and for
commercial entities will require a licence after the initial free trial period on
the web. Full conditions of use will be made available on the web site and
through GeneBio (www.genebio.com), the exclusive worldwide distributor
of GlycoSuiteDB.
P88-S
Disulfide bridge determination of SETI-IIa, a squash
trypsin inhibitor.
V.M. Faca, L.J. Greene; FMRP-USP, Av. Bandeirantes, 3900, Ribeirao Preto,
Sao Paulo 14049-900 Brazil
The squash trypsin inhibitor SETI-IIa, one of the smallest strong inhibitors
described in the literature, contains 31 aminoacids residues of which 6 are
cysteine residues. Its amino acid sequence is: EDRKCPKILMRCKRDSDCLAKC
TCQESGYCG. It forms a compact tridimensional structure maintained by
three disulfide bonds. Due to its small size, this inhibitor can be synthesized,
using Fmoc chemistry. The reoxidation step which requires formation of the
correct disulfide bonds is a critical step in obtaining the active inhibitor and
knowledge of the correct disulfide pairing is required. We describe a procedure
to determine the disulfide pairing of SETI-IIa using small amounts of
protein. About 50 nmol (175 �g) of native SETI was submitted to digestion
with thermolysin for 72 hours at 45�C in a 0.5 M MES buffer pH 6.5. The fragments
obtained were submitted to RP-HPLC in a C18 column (4.6 � 220 mm)
in a TFA/Acetonitrile elution system. The major fragments were collected an
identified by amino acid composition and sequencing by Edman degradation.
The disulfide bridges obtained from native SETI-IIa and their yields were:
Cys1–Cys4 (50%), Cys2–Cys5 (10%) and Cys3–Cys6 (38%). This procedure
will permit us to characterize refolded synthetic analogs of SETI-IIA
Supported by FAPESP. Faça, V.M. has a FAPESP pre-doctoral fellowship.
JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000 209