TY - JOUR
T1 - Development and evaluation of silica-based lectin microcolumns for glycoform analysis of alpha1-acid glycoprotein
AU - Zhang, Chenhua
AU - Hage, David S.
N1 - Funding Information:
This work was supported by the National Institutes of Health under grant R01 GM044931 .
Publisher Copyright:
© 2019 Elsevier B.V.
PY - 2019/10/31
Y1 - 2019/10/31
N2 - Silica-based lectin microcolumns were developed and optimized for the separation and analysis of glycoform fractions in alpha1-acid glycoprotein (AGP) based on both the degree of branching and level of fucosylation. Concanavalin A (Con A) and Aleuria Aurantia lectin (AAL) were immobilized onto HPLC-grade silica by reductive amination and packed into 2.1 mm i.d. × 5.0 cm microcolumns. Factors examined for these microcolumns include their protein content, binding capacity, binding strength and band-broadening under isocratic conditions (Con A) or step elution conditions (AAL) and in the presence of various flow rates or temperatures. These factors were examined by using experiments based on frontal analysis, zonal elution, peak profiling and peak decay analysis. Up to 200 μg AGP could be loaded onto a Con A microcolumn and provide linear elution conditions, and 100 μg AGP could be applied to an AAL microcolumn. The final conditions for separating retained and non-retained AGP glycoform fractions on a Con A microcolumn used a flow rate of 50 μL min−1 and a temperature of 50 °C, which gave a separation of these fractions within 20 min or less. The final conditions for an AAL microcolumn included a flow rate of 0.75 mL min−1, a temperature of 50 °C, and the use of 2.0 mM L-fucose as a competing agent for elution, giving a separation of non-retained and retained AGP glycoforms in 6 min or less. The inter-day precisions were ±0.7–4.0% or less for the retention times of the AGP glycoforms and ±2.2–3.0% or less for their peak areas.
AB - Silica-based lectin microcolumns were developed and optimized for the separation and analysis of glycoform fractions in alpha1-acid glycoprotein (AGP) based on both the degree of branching and level of fucosylation. Concanavalin A (Con A) and Aleuria Aurantia lectin (AAL) were immobilized onto HPLC-grade silica by reductive amination and packed into 2.1 mm i.d. × 5.0 cm microcolumns. Factors examined for these microcolumns include their protein content, binding capacity, binding strength and band-broadening under isocratic conditions (Con A) or step elution conditions (AAL) and in the presence of various flow rates or temperatures. These factors were examined by using experiments based on frontal analysis, zonal elution, peak profiling and peak decay analysis. Up to 200 μg AGP could be loaded onto a Con A microcolumn and provide linear elution conditions, and 100 μg AGP could be applied to an AAL microcolumn. The final conditions for separating retained and non-retained AGP glycoform fractions on a Con A microcolumn used a flow rate of 50 μL min−1 and a temperature of 50 °C, which gave a separation of these fractions within 20 min or less. The final conditions for an AAL microcolumn included a flow rate of 0.75 mL min−1, a temperature of 50 °C, and the use of 2.0 mM L-fucose as a competing agent for elution, giving a separation of non-retained and retained AGP glycoforms in 6 min or less. The inter-day precisions were ±0.7–4.0% or less for the retention times of the AGP glycoforms and ±2.2–3.0% or less for their peak areas.
KW - Affinity microcolumn
KW - Aleuria aurantia lectin
KW - Alpha-acid glycoprotein
KW - Concanavalin A
KW - Glycoform analysis
KW - Lectin affinity chromatography
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U2 - 10.1016/j.aca.2019.05.060
DO - 10.1016/j.aca.2019.05.060
M3 - Article
C2 - 31358219
AN - SCOPUS:85066333725
SN - 0003-2670
VL - 1078
SP - 189
EP - 199
JO - Analytica Chimica Acta
JF - Analytica Chimica Acta
ER -