Analysis of the microheterogeneity of the glycoprotein chorionic gonadotropin with high-performance capillary electrophoresis

Dean E. Morbeck, Benjamin J. Madden, Daniel J Mc Cormick

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

Human chorionic gonadotropin (hCG) is a heteromeric glycoprotein hormone with a molecular mass of ca. 38 000. The carbohydrate side chains terminate with sialic acid and account for roughly 30% of the mass of the hormone. Glycoforms of hCG have been routinely identified with conventional methods of isoelectric focusing or chromatofocusing and exhibit varied bioactivity. In the present report, high-performance capillary electrophoresis (HPCE) was used to separate the glycoforms of hCG and its subunits. Optimal conditions for obtaining near-baseline resolution of the glycoforms were 25 mM borate, pH 8.8 containing 5 mM diaminopropane. The samples were separated in a 100 cm fused-silica capillary with an internal diameter of 50 μm at 25 kV and 28°C. In its native form, hCG migrated in less than 50 min as 8 distinct, highly resolved peaks. In the absence of diaminopropane, hCG migrated as a single, broad peak. When analyzed individually, the α subunit separated into four peaks and the β subunit resolved as seven peaks. The two subunits could also be separated when the heterodimer was incubated in 0.25% trifluoroacetic acid for 1 h prior to injection into the capillary. To illustrate the potential clinical application of this technique, four different sources of hCG were analyzed. The number of different isoforms was constant among the four samples; however, the relative concentration (amounts) of the isoforms varied. These results illustrate the potential utility of HPCE in the clinical diagnostic analysis of hCG microheterogeneity.

Original languageEnglish (US)
Pages (from-to)217-224
Number of pages8
JournalJournal of Chromatography A
Volume680
Issue number1
DOIs
StatePublished - Sep 30 1994

Fingerprint

Capillary electrophoresis
Capillary Electrophoresis
Chorionic Gonadotropin
Glycoproteins
Protein Isoforms
Hormones
Trifluoroacetic Acid
Borates
Isoelectric Focusing
Molecular mass
N-Acetylneuraminic Acid
Fused silica
Bioactivity
Silicon Dioxide
Carbohydrates
Injections

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Analysis of the microheterogeneity of the glycoprotein chorionic gonadotropin with high-performance capillary electrophoresis. / Morbeck, Dean E.; Madden, Benjamin J.; Mc Cormick, Daniel J.

In: Journal of Chromatography A, Vol. 680, No. 1, 30.09.1994, p. 217-224.

Research output: Contribution to journalArticle

@article{5a53b8aa77254ccaac3d27ebd361a31e,
title = "Analysis of the microheterogeneity of the glycoprotein chorionic gonadotropin with high-performance capillary electrophoresis",
abstract = "Human chorionic gonadotropin (hCG) is a heteromeric glycoprotein hormone with a molecular mass of ca. 38 000. The carbohydrate side chains terminate with sialic acid and account for roughly 30{\%} of the mass of the hormone. Glycoforms of hCG have been routinely identified with conventional methods of isoelectric focusing or chromatofocusing and exhibit varied bioactivity. In the present report, high-performance capillary electrophoresis (HPCE) was used to separate the glycoforms of hCG and its subunits. Optimal conditions for obtaining near-baseline resolution of the glycoforms were 25 mM borate, pH 8.8 containing 5 mM diaminopropane. The samples were separated in a 100 cm fused-silica capillary with an internal diameter of 50 μm at 25 kV and 28°C. In its native form, hCG migrated in less than 50 min as 8 distinct, highly resolved peaks. In the absence of diaminopropane, hCG migrated as a single, broad peak. When analyzed individually, the α subunit separated into four peaks and the β subunit resolved as seven peaks. The two subunits could also be separated when the heterodimer was incubated in 0.25{\%} trifluoroacetic acid for 1 h prior to injection into the capillary. To illustrate the potential clinical application of this technique, four different sources of hCG were analyzed. The number of different isoforms was constant among the four samples; however, the relative concentration (amounts) of the isoforms varied. These results illustrate the potential utility of HPCE in the clinical diagnostic analysis of hCG microheterogeneity.",
author = "Morbeck, {Dean E.} and Madden, {Benjamin J.} and {Mc Cormick}, {Daniel J}",
year = "1994",
month = "9",
day = "30",
doi = "10.1016/0021-9673(94)80070-7",
language = "English (US)",
volume = "680",
pages = "217--224",
journal = "Journal of Chromatography",
issn = "0021-9673",
publisher = "Elsevier",
number = "1",

}

TY - JOUR

T1 - Analysis of the microheterogeneity of the glycoprotein chorionic gonadotropin with high-performance capillary electrophoresis

AU - Morbeck, Dean E.

AU - Madden, Benjamin J.

AU - Mc Cormick, Daniel J

PY - 1994/9/30

Y1 - 1994/9/30

N2 - Human chorionic gonadotropin (hCG) is a heteromeric glycoprotein hormone with a molecular mass of ca. 38 000. The carbohydrate side chains terminate with sialic acid and account for roughly 30% of the mass of the hormone. Glycoforms of hCG have been routinely identified with conventional methods of isoelectric focusing or chromatofocusing and exhibit varied bioactivity. In the present report, high-performance capillary electrophoresis (HPCE) was used to separate the glycoforms of hCG and its subunits. Optimal conditions for obtaining near-baseline resolution of the glycoforms were 25 mM borate, pH 8.8 containing 5 mM diaminopropane. The samples were separated in a 100 cm fused-silica capillary with an internal diameter of 50 μm at 25 kV and 28°C. In its native form, hCG migrated in less than 50 min as 8 distinct, highly resolved peaks. In the absence of diaminopropane, hCG migrated as a single, broad peak. When analyzed individually, the α subunit separated into four peaks and the β subunit resolved as seven peaks. The two subunits could also be separated when the heterodimer was incubated in 0.25% trifluoroacetic acid for 1 h prior to injection into the capillary. To illustrate the potential clinical application of this technique, four different sources of hCG were analyzed. The number of different isoforms was constant among the four samples; however, the relative concentration (amounts) of the isoforms varied. These results illustrate the potential utility of HPCE in the clinical diagnostic analysis of hCG microheterogeneity.

AB - Human chorionic gonadotropin (hCG) is a heteromeric glycoprotein hormone with a molecular mass of ca. 38 000. The carbohydrate side chains terminate with sialic acid and account for roughly 30% of the mass of the hormone. Glycoforms of hCG have been routinely identified with conventional methods of isoelectric focusing or chromatofocusing and exhibit varied bioactivity. In the present report, high-performance capillary electrophoresis (HPCE) was used to separate the glycoforms of hCG and its subunits. Optimal conditions for obtaining near-baseline resolution of the glycoforms were 25 mM borate, pH 8.8 containing 5 mM diaminopropane. The samples were separated in a 100 cm fused-silica capillary with an internal diameter of 50 μm at 25 kV and 28°C. In its native form, hCG migrated in less than 50 min as 8 distinct, highly resolved peaks. In the absence of diaminopropane, hCG migrated as a single, broad peak. When analyzed individually, the α subunit separated into four peaks and the β subunit resolved as seven peaks. The two subunits could also be separated when the heterodimer was incubated in 0.25% trifluoroacetic acid for 1 h prior to injection into the capillary. To illustrate the potential clinical application of this technique, four different sources of hCG were analyzed. The number of different isoforms was constant among the four samples; however, the relative concentration (amounts) of the isoforms varied. These results illustrate the potential utility of HPCE in the clinical diagnostic analysis of hCG microheterogeneity.

UR - http://www.scopus.com/inward/record.url?scp=0027965397&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027965397&partnerID=8YFLogxK

U2 - 10.1016/0021-9673(94)80070-7

DO - 10.1016/0021-9673(94)80070-7

M3 - Article

AN - SCOPUS:0027965397

VL - 680

SP - 217

EP - 224

JO - Journal of Chromatography

JF - Journal of Chromatography

SN - 0021-9673

IS - 1

ER -