TY - JOUR
T1 - Glycoform analysis of alpha1-acid glycoprotein based on capillary electrophoresis and electrophoretic injection
AU - Zhang, Chenhua
AU - Bi, Cong
AU - Clarke, William
AU - Hage, David S.
N1 - Funding Information:
This work was supported by the National Institutes of Health under grant R01 GM044931 . Appendix A
PY - 2017/11/10
Y1 - 2017/11/10
N2 - A method based on capillary electrophoresis (CE) with electrophoretic injection and absorbance detection was developed for the direct analysis of AGP glycoforms in human serum. Electrophoretic injection of AGP was performed in the reversed-polarity mode of CE with a capillary coated with poly(ethylene oxide) and that had minimal electroosmotic flow. This situation created an essentially stationary interface between the sample and running buffer during injection and sample stacking. This approach allowed an 11,000-fold increase in sample loading for a 5 min injection versus hydrodynamic injection and without introducing any significant levels of extra band-broadening. This method was used with sample pretreatment methods based on acid precipitation and desalting to examine AGP glycoforms in only 65 μL of serum. A limit of detection of 2.1–11.3 nM was obtained for the major AGP glycoform bands in serum, and the sample pretreatment method gave a recovery of 72.3–80.9% for these glycoforms. The precision for the migration times was ± 0.08–0.13% and the precision for the peak areas was ± 0.34–1.18% when using serum samples and an internal standard. This method was used for both normal pooled serum and serum from individuals with systemic lupus erythematosus. Results were obtained in a separation time of 25 min and allowed the comparison of up to eleven glycoform bands in these samples. A similar approach may be useful in examining additional glycoproteins in serum or other types of biological samples.
AB - A method based on capillary electrophoresis (CE) with electrophoretic injection and absorbance detection was developed for the direct analysis of AGP glycoforms in human serum. Electrophoretic injection of AGP was performed in the reversed-polarity mode of CE with a capillary coated with poly(ethylene oxide) and that had minimal electroosmotic flow. This situation created an essentially stationary interface between the sample and running buffer during injection and sample stacking. This approach allowed an 11,000-fold increase in sample loading for a 5 min injection versus hydrodynamic injection and without introducing any significant levels of extra band-broadening. This method was used with sample pretreatment methods based on acid precipitation and desalting to examine AGP glycoforms in only 65 μL of serum. A limit of detection of 2.1–11.3 nM was obtained for the major AGP glycoform bands in serum, and the sample pretreatment method gave a recovery of 72.3–80.9% for these glycoforms. The precision for the migration times was ± 0.08–0.13% and the precision for the peak areas was ± 0.34–1.18% when using serum samples and an internal standard. This method was used for both normal pooled serum and serum from individuals with systemic lupus erythematosus. Results were obtained in a separation time of 25 min and allowed the comparison of up to eleven glycoform bands in these samples. A similar approach may be useful in examining additional glycoproteins in serum or other types of biological samples.
KW - Alpha-acid glycoprotein
KW - Capillary electrophoresis
KW - Electrophoretic injection
KW - Glycoform analysis
KW - Sample stacking
KW - Systemic lupus erythematosus
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U2 - 10.1016/j.chroma.2017.08.032
DO - 10.1016/j.chroma.2017.08.032
M3 - Article
C2 - 28844299
AN - SCOPUS:85027986072
VL - 1523
SP - 114
EP - 122
JO - Journal of Chromatography A
JF - Journal of Chromatography A
SN - 0021-9673
ER -