4 research outputs found
Heparin Isomeric Oligosaccharide Separation Using Volatile Salt Strong Anion Exchange Chromatography
The
complexity of heparin and heparan sulfate saccharides makes
their purification, including many isomeric structures, very challenging
and is a bottleneck for structure–activity studies. High-resolution
separations have been achieved by strong anion exchange (SAX) chromatography
on Propac PA1 and cetyltrimethylammonium (CTA)-C<sub>18</sub> silica
columns; however, these entail subsequent desalting methodologies
and consequent sample losses and are incompatible with orthogonal
chromatography methodologies and, in particular, mass spectrometry.
Here, we present the CTA-SAX purification of heparin oligosaccharides
using volatile salt (VS) buffer. In VSCTA-SAX, the use of ammonium
bicarbonate buffer for elution improves resolution through both weaker
dissociation and conformational coordination of the ammonium across
the sulfate groups. Using ion mobility mass spectrometry, we demonstrate
that isomeric structures have different structural conformations,
which makes chromatographic separation achievable. Resolution of such
structures is improved compared to standard SAX methods, and in addition,
VSCTA-SAX provides an orthogonal method to isolate saccharides with
higher purity. Because ammonium bicarbonate is used, the samples can
be evaporated rather than desalted, preventing substantial sample
loss and allowing more effective subsequent analysis by electrospray
mass spectrometry. We conclude that VSCTA-SAX is a powerful new tool
that helps address the difficult challenge of heparin/heparan sulfate
saccharide separation and will enhance structure–activity studies
Predicting Structural Motifs of Glycosaminoglycans using Cryogenic Infrared Spectroscopy and Random Forest
In recent years, glycosaminoglycans (GAGs) have emerged
into the
focus of biochemical and biomedical research due to their importance
in a variety of physiological processes. These molecules show great
diversity, which makes their analysis highly challenging. A promising
tool for identifying the structural motifs and conformation of shorter
GAG chains is cryogenic gas-phase infrared (IR) spectroscopy. In this
work, the cryogenic gas-phase IR spectra of mass-selected heparan
sulfate (HS) di-, tetra-, and hexasaccharide ions were recorded to
extract vibrational features that are characteristic to structural
motifs. The data were augmented with chondroitin sulfate (CS) disaccharide
spectra to assemble a training library for random forest (RF) classifiers.
These were used to discriminate between GAG classes (CS or HS) and
different sulfate positions (2-O-, 4-O-, 6-O-, and N-sulfation). With
optimized data preprocessing and RF modeling, a prediction accuracy
of >97% was achieved for HS tetra- and hexasaccharides based on
a
training set of only 21 spectra. These results exemplify the importance
of combining gas-phase cryogenic IR ion spectroscopy with machine
learning to improve the future analytical workflow for GAG sequencing
and that of other biomolecules, such as metabolites
Table_1_Role of N-Glycosylation in FcγRIIIa interaction with IgG.xlsx
Immunoglobulins G (IgG) and their Fc gamma receptors (FcγRs) play important roles in our immune system. The conserved N-glycan in the Fc region of IgG1 impacts interaction of IgG with FcγRs and the resulting effector functions, which has led to the design of antibody therapeutics with greatly improved antibody-dependent cell cytotoxicity (ADCC) activities. Studies have suggested that also N-glycosylation of the FcγRIII affects receptor interactions with IgG, but detailed studies of the interaction of IgG1 and FcγRIIIa with distinct N-glycans have been hindered by the natural heterogeneity in N-glycosylation. In this study, we employed comprehensive genetic engineering of the N-glycosylation capacities in mammalian cell lines to express IgG1 and FcγRIIIa with different N-glycan structures to more generally explore the role of N-glycosylation in IgG1:FcγRIIIa binding interactions. We included FcγRIIIa variants of both the 158F and 158V allotypes and investigated the key N-glycan features that affected binding affinity. Our study confirms that afucosylated IgG1 has the highest binding affinity to oligomannose FcγRIIIa, a glycan structure commonly found on Asn162 on FcγRIIIa expressed by NK cells but not monocytes or recombinantly expressed FcγRIIIa.</p
DataSheet_1_Role of N-Glycosylation in FcγRIIIa interaction with IgG.pdf
Immunoglobulins G (IgG) and their Fc gamma receptors (FcγRs) play important roles in our immune system. The conserved N-glycan in the Fc region of IgG1 impacts interaction of IgG with FcγRs and the resulting effector functions, which has led to the design of antibody therapeutics with greatly improved antibody-dependent cell cytotoxicity (ADCC) activities. Studies have suggested that also N-glycosylation of the FcγRIII affects receptor interactions with IgG, but detailed studies of the interaction of IgG1 and FcγRIIIa with distinct N-glycans have been hindered by the natural heterogeneity in N-glycosylation. In this study, we employed comprehensive genetic engineering of the N-glycosylation capacities in mammalian cell lines to express IgG1 and FcγRIIIa with different N-glycan structures to more generally explore the role of N-glycosylation in IgG1:FcγRIIIa binding interactions. We included FcγRIIIa variants of both the 158F and 158V allotypes and investigated the key N-glycan features that affected binding affinity. Our study confirms that afucosylated IgG1 has the highest binding affinity to oligomannose FcγRIIIa, a glycan structure commonly found on Asn162 on FcγRIIIa expressed by NK cells but not monocytes or recombinantly expressed FcγRIIIa.</p