Clinical Chemistry 46: 1796-1803, 2000;
(Clinical Chemistry. 2000;46:1796-1803.)
© 2000 American Association for Clinical Chemistry, Inc.
Determination of the Glycoforms of Human Chorionic Gonadotropin ß-Core Fragment by Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
Eli S. Jacoby1,
Andrew T. Kicman2,
Paul Laidler2 and
Ray K. Iles1,a
1
The Williamson Laboratory, Department of Obstetrics and Gynaecology, St. Bartholomew’s & the Royal London School of Medicine and Dentistry, West Smithfield, London EC1A 7BE, United Kingdom.
2
The Drug Control Centre, King’s College London,
Franklin-Wilkins Building, 150 Stamford St., London SE1 8WA, United
Kingdom.
a Author for correspondence. Fax 44-020-7601-7050; e-mail r.k.iles{at}mds.qmw.ac.uk
 |
Abstract
|
|---|
Background: Metabolism of human chorionic gonadotropin (hCG) in
the serum and kidney yields the terminal urinary product hCG ß-core
fragment (hCGßcf), comprising two disulfide-linked peptides
(ß6-ß40 and ß55-ß92) of which one (ß6-ß40) retains
truncated N-linked sugars. Hyperglycosylated hCGßcf may indicate
choriocarcinoma or Down syndrome, but the glycosylation profile of
hCGßcf has not been thoroughly evaluated.
Methods: hCGßcf, purified from pregnancy urine, was reduced by
"on-target" dithiothreitol (DTT) reduction and analyzed by
matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF MS). The mass ([M+H]+)
of the primary sequence of the glycosylated peptide ß6-ß40 was
subtracted from the m/z values of the discrete peaks
observed to give the masses of the carbohydrate moieties. Carbohydrate
structure was predicted by sequentially subtracting the masses of the
monosaccharide residues corresponding to N-linked carbohydrates of the
hCG ß-subunit reported in the literature.
Results: Mass spectra of hCGßcf revealed a broad triple peak at
m/z 8700–11300. After reduction, the triple peak was
replaced by a discrete set of peaks between m/z 4156 and
6354. A peak at m/z 4156.8 corresponded to the
nonglycosylated peptide (ß55-ß92). The remaining nine peaks
indicated that urinary hCGßcf comprises a set of glycoforms smaller
and larger than the trimannosyl core.
Conclusions: hCGßcf comprises a wider set of glycoforms than
reported previously. Peaks of highest mass indicate evidence of
hyperglycosylated carbohydrate moieties. The data support previous
reports that hCGßcf oligosaccharides lack sialic acid and galactose
residues. No indication was found of a ß6-ß40 peptide that was
entirely devoid of carbohydrate.
|
Introduction
|
|---|
The heterodimeric placental hormone human chorionic gonadotropin
(hCG)1
is a widely studied member of the glycoprotein hormone family
that includes luteinizing hormone, thyroid-stimulating
hormone, and follicle-stimulating hormone. Each consists of a common
subunit and a unique ß subunit that confers specific hormonal
activity. hCG sustains early pregnancy by binding luteinizing
hormone receptors at the corpus luteum surface and stimulating
progesterone secretion until the placenta is able to carry out this
function itself. In addition to being universally used as an early
marker of pregnancy, hCG is monitored in sports drug testing
(1). The hCG ß-subunit (hCGß) is also a marker of
various epithelial and gestational trophoblast carcinomas
(2)(3), where it may exert independent
stimulatory activity (4)(5). The precise
structure of hCG has been well characterized by HPLC-mass spectrometry
(6)(7) and by crystallographic analysis
(8)(9). Peptide mass mapping by matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS), using trypsin for digestion, has also been found to be
a suitable technique for the identification of hCG and its subunits
(10)(11).
hCG, in common with other glycoprotein hormones, is characterized by
sugar moieties on both subunits that determine receptor affinity and
metabolic clearance. The currently accepted structural model of the
N-linked carbohydrates found on hCGß is of a complex biantennary
moiety terminating in sialic acid based on a trimannosyl core
(12). Despite the model, wide carbohydrate heterogeneity has
been found. Mono-, bi-, and triantennary N-linked sugars occur in
hCGß in both normal and abnormal pregnancies as well as in
choriocarcinomas (12)(13)(14)(15)(16)(17). An increase in the triantennary
form seems to be a feature indicative of an abnormal state and may be
of diagnostic value. In normal pregnancies, Elliott et al.
(14) found that the biantennary form accounted for 76.5% of
the N-linked oligosaccharide content of urinary hCGß, that 21.6% was
triantennary, and that the remainder was monoantennary (Fig. 1
). In gestational choriocarcinoma, by contrast, the triantennary
content accounted for up to 63.6% of the total. A similar
increase in hCG hyperglycosylation has been detected in Down syndrome
by Cole et al. (18). Indeed, an ELISA for hyperglycosylated
hCG has been developed based on a monoclonal antibody raised to the
hyperglycosylated variant (19)(20). Prenatal
diagnostic testing of maternal urine based on this ELISA is claimed to
detect up to 97% of Down syndrome cases with a 5% false-positive rate
when modeled with values of other markers (21).
View larger version (22K):
[in this window]
[in a new window]
|
Figure 1. Structures of the asialo asparagine-linked
oligosaccharides of the hCGß subunit from normal pregnancy,
hydatidiform mole, choriocarcinoma (I,
III, and IV, respectively), and
gestational diabetes (II).
Adapted from Elliott et al. (14).
|
|
Similarly, carbohydrate heterogeneity remains in the terminal urinary
degradation product of the ß-subunit, the hCG ß-core fragment
(hCGßcf). This core fragment has a primary structure of 73 amino
acids, consisting of two polypeptides: ß6-ß40 and ß55-ß92,
joined by four cystine residues (Fig. 2
). Only the ß6-ß40 chain is glycosylated, retaining the two
complex N-glycans linked to asparagine residues 13 and 30. However, as
a consequence of metabolism, these branched chain carbohydrates are
truncated. Various studies of the carbohydrate structure of hCGßcf
using a combination of immunoaffinity, gel-filtration, and ion-exchange
chromatography, glycosidase digestion, hydrazinolysis, and periodate
oxidation have been reported (22)(23)(24)(25)(26). A population of
shortened mono- and biantennary carbohydrate cores has been suggested,
which may or may not be fucosylated (22). There is
disagreement over whether these moieties possess sialic acid or
galactose residues, although there is some evidence that they do extend
beyond the trimannosyl cores (24). In an attempt to help
resolve this disagreement, we investigated the structural variation in
these oligosaccharide moieties, using an alternative approach that
incorporates analysis by MALDI-TOF MS.
View larger version (11K):
[in this window]
[in a new window]
|
Figure 2. Schematic diagram of the primary amino acid sequence of
hCGßcf, showing positions of disulfide bonds and N-linked
carbohydrates.
|
|
MALDI-TOF MS is a powerful tool for determining the mass of
macromolecules, as first reported by Karas and Hillenkamp
(27), yielding signals that correspond mainly to singly
charged molecular ions. Chemical or enzymatic cleavage of
posttranslationally modified proteins, followed by MALDI-TOF MS
analysis, has been used for the purpose of structural characterization.
Exoglycosidase digestion with MALDI-TOF MS analysis has been used to
elucidate carbohydrate sequences and composition of glycoproteins
(28). Nonetheless, at first glance, this approach
appears to be overcomplicated for investigating the glycoforms of
hCGßcf, particularly because the branch chain sequence is unlikely to
be affected by metabolism of the ß-subunit, albeit that the chain is
truncated. Indeed, exoglycosidase digestion could confound
interpretation of results because absolute specificity for one
substrate is rare and this may possibly lead to the presence of
artifacts. However, direct analysis of hCGßcf by low-resolution
MALDI-TOF MS is not sufficiently discriminating to identify individual
glycoforms because of the lack of resolution between them. A simple but
potentially useful approach could be to reduce the disulfide bonds,
e.g., with DTT, to yield the two separate polypeptide chains before
analysis. Separation of the ß55-ß92 chain, which is of no
diagnostic value, from the ß6-ß40 chain is advantageous because the
overall masses of the glycoproteins being analyzed are reduced, which
is conducive to discriminating between them. The mass of each
glycosylation variant on the ß6-ß40 chain can then be determined by
calculating the difference in mass between each peak observed to the
mass of its corresponding primary amino acid sequence, i.e.,
nonglycosylated ß6-ß40. The presence of residual triantennary
sugars on hCGßcf has not been as well determined as for free hCGß
but should be characterized to determine any diagnostic potential. In
this study, we used MALDI-TOF MS, for the first time, to directly
observe and determine the number of glycoforms in a purified sample of
pooled pregnancy hCGßcf.
|
Materials and Methods
|
|---|
materials
The hCGßcf used in this study was isolated from crude urinary
hCG (Sigma) and characterized previously (29). DTT, for the
reduction of disulfide bonds, and
NH4HCO3 were obtained from
Sigma-Aldrich. The MALDI-TOF MS matrix 3,5-dimethoxy-4-hydroxycinnamic
acid (sinapinic acid; SA) was also purchased from Sigma-Aldrich and
used without further purification. Acetonitrile and trifluoroacetic
acid (TFA) were of analytical grade and obtained from Merck.
maldi-tof ms
To acquire spectra of the nonreduced molecule, we applied 0.5 µL
of hCGßcf [20 µmol/L in distilled, deionized
H2O (dH2O)] to a stainless
steel MALDI-TOF MS target and mixed it with 1.0 µL of SA [20 g/L in
70:30 (by volume) acetonitrile-1 mL/L TFA in
dH2O] and air dried. The instrument was
calibrated externally using horse heart cytochrome C
(Mr 12 360.1; Sigma-Aldrich). Spectra
of reduced glycoforms were obtained by first mixing, on a MALDI-TOF MS
target, 0.5 µL of hCGßcf (20 µmol/L in
dH2O) with 0.5 µL of DTT (100 mmol/L in 100
mmol/L NH4HCO3, pH 8.8)
(30). When this mixture was virtually dry, 0.5 µL of 1
mL/L TFA in dH2O was added to the remaining
droplet. After the mixture was again allowed to partially dry, 1.0 µL
of SA [20 g/L in a 70:30 mixture (by volume) of acetonitrile-1 mL/L
TFA in dH2O] was added, and the sample was
air-dried as before. Mass spectrometric analysis was carried out on VG
TofSpec E (Micromass) and LaserMAT 2000 (Finnigan MAT) instruments,
with 1.5 m and 0.5 m flight tubes, respectively, and both
operating at an accelerating voltage of 20 kV. A pulsed nitrogen laser
(
max = 337 nm) was used to desorb ions from
the samples, which were detected by a microchannel plate detector at a
sampling rate of 500 MHz. Spectra were generated by summing 30–40
laser shots. Data were acquired from both instruments operating in the
positive linear mode.
treatment of results
Initially, the peak relating to the nonglycosylated hCGßcf
peptide (ß55-ß92) was internally calibrated to the average
molecular mass of its given primary sequence,
[M+H]+ 4156.8 (calculated using the Protein
Abacus computer software; Finnigan MAT). This facilitated the automatic
correction of the mass values of the remaining peaks to their correct
values.
To determine the inferred mass values of the hCGßcf carbohydrate
moieties, we subtracted the average molecular mass
([M+H]+ 3752.4) of the primary
sequence of the glycosylated hCGßcf polypeptide (ß6-ß40) from the
mass ([M+H]+) values of the acquired peaks
(Table 1
). The carbohydrate content of these peaks was then deduced by
the sequential subtraction of the masses of the sugar residues present
in hCG ß-subunit carbohydrates (Table 2
). Carbohydrate structures (Table 3
) could then be predicted from the deduced monosaccharide
content, taking into account the very small difference between the
calculated and the observed masses (
0.15%). Different combinations
of carbohydrate structures are shown where the mass of a peak indicated
several monosaccharides from which several permutations could be
construed. Although this study did not yield any conformational
information, the anomeric glycosidic linkages within the carbohydrate
structures in Table 3
have been described previously (14)
and were used in our analysis.
View this table:
[in this window]
[in a new window]
|
Table 2. Relative molecular mass (RMM) of the monosaccharides
expected to be present in hCGß subunit N-linked
carbohydrates.
|
|
View this table:
[in this window]
[in a new window]
|
Table 3. Proposed structures of the oligosaccharides in the
glycoforms contributing to the microheterogeneity of
hCGßcf.1
|
|
|
Results
|
|---|
SA was selected as the MALDI-TOF MS matrix of choice for this
investigation because other standard matrices used in protein analysis,
-cyano-4-hydoxycinnamic acid and sDHB [a 9:1 (by weight) mixture of
2,5-dihydroxybenzoic acid and 5-methoxysalicylic acid], did not yield
intense spectra for hCGßcf (data not shown). A typical spectrum of
hCGßcf obtained by MALDI-TOF MS using SA is shown in Fig. 3
. The spectrum is characterized by a broad triple peak at
m/z 8700–11300 with discernible peaks at m/z
9110, 9930, and 10770. After reduction with DTT (Fig. 4
), the triple peak disappeared and was replaced by a set of
resolved peaks between m/z 4156 and 6354. The initial peak
(Fig. 4
), at m/z 4164.2, was presumed to arise from the
nonglycosylated hCGßcf peptide chain (ß55-ß92) because the
m/z value is within the 0.5% error allowed for the linear
mode of the MALDI instrument. To obtain corrected values for the
remaining peaks, this peak was internally calibrated to the average
molecular mass of the given primary sequence,
[M+H]+ 4156.8 (calculated using the Protein
Abacus computer software). Despite the fact that hCGßcf
asparagine-linked carbohydrate moieties were not observed directly, the
low percentage errors between the observed and the expected mass values
of the peaks acquired show that it is likely that real glycoforms were
detected (Table 1
).
View larger version (12K):
[in this window]
[in a new window]
|
Figure 3. MALDI-TOF mass spectrum of purified hCGßcf in SA matrix
generated by summation of the signals from 40 laser shots and
calibrated externally using horse heart cytochrome C
(Mr 12 360.1).
|
|
View larger version (17K):
[in this window]
[in a new window]
|
Figure 4. MALDI-TOF mass spectrum of hCGßcf after "on-target"
DTT reduction and using SA matrix.
The spectrum was generated by summation of the signals from 30 laser
shots and calibrated internally using fragment mass
[M+H]+ 4156.8. Peaks 1–9 are
described in the text.
|
|
Carbohydrate moieties, shown in Table 3
, were determined as described
above. Peak 1 (m/z 4630.2) arises from a polypeptide
possessing a single truncated and fucosylated monoantennary
oligosaccharide
(Man2GlcNAc2Fuc) with a
predicted mass of 876.8, whereas the polypeptide giving rise to peak 2
(m/z 4819.6) has two shortened fucosylated monoantennary
sugars (ManGlcNAc2Fuc and
GlcNAc2Fuc) with a predicted mass of 1064.0.
Peaks 6 (m/z 5677.9) and 7 (m/z 5821.1)
apparently arise from hCGßcf glycoforms containing two N-linked
trimannosyl core carbohydrates
(Man3GlcNAc2). In peak 6, a
single trimannosyl core possesses fucose (with a predicted mass of
1931.8), whereas both are fucosylated in peak 7 (with a predicted mass
of 2077.9). The combination of monosaccharide residues (with a
predicted mass of 2420.3) that make up the glycoform observed at
peak 8 (m/z 6179.0) can be configured to produce an
identical pair of sugar structures that are shortened versions
(GlcNAc2Man2GlcNAc2)
of carbohydrate structures II or IV found by Elliott et al.
(14). The number of monosaccharide residues calculated for
the polypeptides giving mass spectral peaks 3–5 and peak 9 precludes
the determination of a single pair of glycoforms, and for these
alternative structures have been suggested. The predicted mass of
1242.2 for the carbohydrate moiety of the glycoform at peak 3
(m/z 5000.8) indicates either a single fucosylated
trimannosyl core attached to a monoantennary
N-acetylglucosamine
(Man3GlcNAc3Fuc) or a
trimannosyl core
(Man3GlcNAc2) at one
glycosylation site and a single GlcNAc at the other, one of which is
fucosylated. Peak 4 (m/z 5358.2) indicates a glycoform with
a predicted carbohydrate mass of 1607.5, which can translate to two
shortened core sugars
(Man2GlcNAc2) differing by
a fucose residue. An alternative combination includes a trimannosyl
core and a shortened core (ManGlcNAc2), one of
which is fucosylated. Similarly, the predicted carbohydrate mass
(1769.6) for the glycoform seen at peak 5 (m/z 5517.6)
indicates a monosaccharide content from which may be constructed a
trimannosyl core
(Man3GlcNAc2) and a
truncated core oligosaccharide
(Man2GlcNAc2), one of which
is connected to a fucose residue. Finally, two extended trimannosyl
cores (Man3GlcNAc4) can be
inferred from a predicted carbohydrate mass of 2598.4 of the
glycopeptide at peak 9 (m/z 6353.8). Structures construed
from this mass may be pairs or a combination of truncated
oligosaccharides II, III, or IV in Fig. 1
. There is an additional peak
in the mass spectra of DTT-reduced hCGßcf between peaks 3 and 4 that
we assume to be an additional glycoform but for which the data analysis
software was unable to acquire a value.
|
Discussion
|
|---|
Direct carbohydrate analyses of hCGßcf in previous studies have
generally suggested that the metabolite retains shortened hCGß
asparagine-linked oligosaccharides that have been trimmed back to their
trimannosyl cores and smaller sugars (22)(23)(24)(25)(26). In addition,
de Medeiros et al. (24) found that 22–44% of hCGßcf
molecules were not bound by the lectin concanavalin A, and of those
88% were completely deprived of any carbohydrate content. After DTT
reduction, our MALDI-TOF MS data indicated the presence of only one
nonglycosylated peptide at m/z 4156.8 within the
dipeptide molecule. If the usually glycosylated peptide
(ß6-ß40) had been completely lacking in carbohydrate, then a peak
at m/z 3752.4 should have been apparent in the mass spectrum
of reduced hCGßcf. This peak was not observed at all in any of the
spectra acquired from reduced hCGßcf. Additionally, our MALDI
analysis of hCGßcf confirms the lack of sialic acid as expected but
also the lack of any galactose in this preparation.
The galactose content of hCGßcf has been reported differently. Direct
carbohydrate analyses after acid hydrolysis (24) or
conversion of sugars to glycamines (23) has detected small
amounts of galactose. Conversely, indirect analysis has found hCGßcf
N-linked sugars lacking in the monosaccharide
(22)(25). The observation that hCGßcf is not
bound by galactose-specific Ricinus communis
agglutinin-agarose affinity matrix (23)(25) has
been attributed to the inability of the matrix to bind one galactose
residue per N-linked sugar, whereas contaminating glycoprotein is
thought to account for hCGßcf galactose detected directly
(22). This direct MALDI-TOF MS analysis indicated that no
galactose was present in the N-linked sugars of the sample we
investigated. It is uncertain where the degradation of hCG
carbohydrates occurs. In their study of hCGß separated from intact
urinary hCG, Liu and Bowers (7) detected a range of both
sialated and asialo mono-, bi-, and triantennary N-linked
oligosaccharides. It would therefore seem likely that the majority of
sialic acid and galactose residues are lost as hCG is processed to
hCGßcf after renal reabsorption. Whether or not this is the case, our
results indicate that hCGßcf possesses neither sialic acid nor
galactose residues. In fact, the glycoforms that appear to be present
in hCGßcf range from a single GlcNAc residue to more complex
structures extending beyond the trimannosyl core. The largest
carbohydrate structures inferred by our results, peaks 8 and 9 in Fig. 4
, may arise from degraded forms of the sugars II and IV in Fig. 1
, determined by Elliott et al. (14) in hCGß.
Absolute quantification of the relative amounts of each N-linked
carbohydrate moiety was not possible using this method.
Semiquantification of relative amounts of glycoform pairs was possible
by determining the areas under the peaks of the reduced peptides.
Unfortunately, many peaks could have arisen from multiple combinations
of sugars. However, the monosaccharides determined from peak 8 (see
Table 3
) give rise to a combination of only two carbohydrates moieties
that may arise from shortened triantennary oligosaccharides. This peak
comprised 15.6% of the carbohydrate content of this sample, which is
in agreement with the triantennary content of normal pregnancy hCG
ß-subunits as determined by Elliott et al. (14). Because
the crude preparation from which this hCGßcf was prepared may have
contained urinary hCGß from not necessarily normal pregnancies, it is
impossible to say whether the larger sugar moieties we observed
originated from either the triantennary carbohydrate (IV in Fig. 1
)
seen in normal or aberrant pregnancies or the biantennary sugar (II in
Fig. 1
) observed almost exclusively in gestational diabetes
(14).
In conclusion, the percentage abundance of other glycoforms
detected by MALDI-TOF MS indicates that it is likely that the
microheterogeneity of the hCGß N-linked carbohydrates found by
Elliott et al. (14) is conserved in hCG ß-core fragments
from all normal and aberrant pregnancies. Although not precisely
defining hCGßcf glycosylation, the MALDI-TOF mass spectrum of
DTT-reduced hCGßcf produces what appears to be a distinctive
"fingerprint". For example, peaks of higher mass attributable to
the increased triantennary sugar content of hCGßcf would be expected
to increase in area in the spectra of hCG ß-core fragments from
individuals with abnormal or Down syndrome pregnancies and
choriocarcinoma. Until now, definitive testing for hyperglycosylation
in hCG in patient urine has required multiple precipitation and
chromatographic steps (31) followed by thiol reduction,
glycan release, and HPLC (14). This is an approach that may
be considered too time-consuming for routine clinical biochemistry.
Although an extract of hCGßcf was analyzed in our current
investigation, MALDI-TOF MS analysis of unextracted urine samples from
pregnant women shows a broad peak corresponding to protonated hCGßcf,
indicating that its concentration in pregnancy urine is sufficiently
high for detection purposes. Further study will help to elucidate
whether the reductive technique described here can be applied to
pregnancy or choriocarcinoma urine, possibly after desalting and
concentration by centrifugal ultrafiltration (10). The
potential clinical utility for distinguishing between normal pregnancy
and Down syndrome pregnancy by this approach needs to be demonstrated
and is currently under investigation.
|
Acknowledgments
|
|---|
This ongoing project is funded by the Joint Research Board of St.
Bartholomew’s Hospital, London.
|
Footnotes
|
|---|
1 Nonstandard abbreviations: hCG, human chorionic gonadotropin; hCGß, hCG ß-subunit; DTT, dithiothreitol; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; hCGßcf, hCG ß-core fragment; SA, sinapinic acid; TFA, trifluoroacetic acid; dH2O, distilled, deionized water; and GlcNAc, N-acetylglucosamine. 
|
References
|
|---|
-
Laidler P, Cowan DA, Hider RC, Kicman AT. New decision limits and quality-control material for detecting human chorionic gonadotropin misuse in sports. Clin Chem 1994;40:1306-1311.[Abstract/Free Full Text]
-
Cole LA, Wang Y, Elliott M, Latif M, Chambers JT, Chambers SK, Schwartz PE. Urinary human chorionic gonadotropin free ß-subunit and core fragment: a new marker of gynecological cancers. Cancer Res 1988;48:1356-1360.[Abstract/Free Full Text]
-
Iles RK, Chard T. Immunochemical analysis of the human chorionic gonadotrophin-like material secreted by ‘normal’ and neoplastic urothelial cells. J Mol Endocrinol 1989;2:107-112.[Abstract/Free Full Text]
-
Gillott DJ, Iles RK, Chard T. The effects of ßhCG on the in vitro growth of bladder cancer cells. Br J Cancer 1996;73:323-326.[ISI][Medline]
[Order article via Infotrieve]
-
Butler SA, Ikram MS, Mathieu S, Iles RK. The increase in bladder carcinoma cell population induced by the free beta subunit of human chorionic gonadotrophin is a result of an anti-apoptosis effect and not cell proliferation. Br J Cancer 2000;82:1553-1556.[ISI][Medline]
[Order article via Infotrieve]
-
Liu C, Bowers LD. Mass spectrometric characterization of the beta-subunit of human chorionic gonadotropin. J Mass Spectrom 1997;32:33-42.[ISI][Medline]
[Order article via Infotrieve]
-
Liu C, Bowers LD. Mass spectrometric characterization of nicked fragments of the ß-subunit of human chorionic gonadotrophin. Clin Chem 1997;43:1172-1181.[Abstract/Free Full Text]
-
Lapthorn AJ, Harris DC, Littlejohn A, Lustbader JW, Canfield RE, Machin KJ, et al. Crystal structure of human chorionic gonadotropin. Nature 1994;369:455-461.[Medline]
[Order article via Infotrieve]
-
Wu H, Lustbader JW, Liu Y, Canfield RE, Hendrickson WA. Structure of human chorionic gonadotropin at 2.6 Å resolution from MAD analysis of the selenomethionyl protein. Structure 1994;2:545-558.[Medline]
[Order article via Infotrieve]
-
Laidler P, Cowan DA, Hider RC, Keane A, Kicman AT. Tryptic mapping of human chorionic gonadotropin by matrix-assisted laser desorption/ionisation mass spectrometry. Rapid Commun Mass Spectrom 1995;9:1021-1026.[ISI][Medline]
[Order article via Infotrieve]
-
Liu C, Bowers LD. Immunoaffinity trapping of urinary human chorionic gonadotropin and its high-performance liquid chromatographic-mass spectrometric confirmation. J Chromatogr B Biomed Appl 1996;687:213-220.[Medline]
[Order article via Infotrieve]
-
Kessler MJ, Reddy MS, Shah RH, Bahl OP. Structures of N-glycosidic carbohydrate units of human chorionic gonadotropin. J Biol Chem 1979;254:7901-7908.[Free Full Text]
-
Damm JBL, Voshol H, Hard K, Kamerling JP, Van Dedem GWK, Vliegenthart JFG. The ß-subunit of human chorionic gonadotropin contains N-glycosidic trisialo tri-antennary and tri’-antennary carbohydrate chains. Glycoconjugate J 1988;5:221-233.
-
Elliot MM, Kardana A, Lustbader JW, Cole LA. Carbohydrate and peptide structure of the - and ß-subunits of human chorionic gonadotropin from normal and aberrant pregnancy and choriocarcinoma. Endocrine 1997;7:15-32.[ISI][Medline]
[Order article via Infotrieve]
-
Kobata A. Altered glycosylation of human chorionic gonadotropin in malignant trophoblastic diseases. Mochizuki M Hussa R eds. Placental protein hormones 1988:133-142 Elsevier Amsterdam. .
-
Mizuochi T, Nishimura R, Derappe C, Taniguchi T, Hamamoto T, Mochizuki M, Kobata A. Structures of the asparagine-linked sugar chains of human chorionic gonadotropin produced in choriocarcinoma. J Biol Chem 1983;258:14126-14129.[Abstract/Free Full Text]
-
Yoshimoto Y, Wolfsen AR, Odell WD. Glycosylation, a variable in hCG production by cancers. Am J Med 1979;67:414-420.[ISI][Medline]
[Order article via Infotrieve]
-
Cole LA, Cermik D, Bahado-Singh R. Oligosaccharide variants of hCG-related molecules: potential screening markers for Down syndrome [Letter]. Prenat Diagn 1997;17:1187-1190.[ISI][Medline]
[Order article via Infotrieve]
-
Birken S, Krichevsky A, O’Connor J, Schlatterer J, Cole L, Kardana A, Canfield R. Development and characterization of antibodies to a nicked and hyperglycosylated form of hCG from a choriocarcinoma patient. Endocrine 1999;10:137-144.[ISI][Medline]
[Order article via Infotrieve]
-
Cole LA, Omrani A, Cermik D, Bahado-Singh RO, Mahoney MJ. Hyperglycosylated hCG, a potential alternative to hCG in Down syndrome screening. Prenat Diagn 1998;18:926-933.[ISI][Medline]
[Order article via Infotrieve]
-
Cole LA, Shahabi S, Oz UA, Rinne KM, Omrani A, Bahado-Singh RO, Mahoney MJ. Urinary screening tests for fetal Down syndrome. II. Hyperglycosylated hCG. Prenat Diagn 1999;19:351-359.[ISI][Medline]
[Order article via Infotrieve]
-
Endo T, Nishimura R, Saito S, Kanazawa K, Numura K, Katsuno M, et al. Carbohydrate structures of ß-core fragment of human chorionic gonadotropin isolated from a pregnant individual. Endocrinology 1992;130:2052-2058.[Abstract]
-
Birken S, Armstrong EG, Kolks MAG, Cole LA, Agosto GM, Krichevsky A, et al. Structure of the human chorionic gonadotropin ßsubunit fragment from pregnancy urine. Endocrinology 1988;123:572-583.[Abstract]
-
de Medeiros SF, Amato F, Matthews CD, Norman RJ. Molecular heterogeneity of the ß-core fragment of human chorionic gonadotrophin. J Endocrinol 1993;139:519-532.[Abstract/Free Full Text]
-
Blithe DL, Akar AH, Wehmann RE, Nisula BC. Purification of ß-core fragment from pregnancy urine and demonstration that its carbohydrate moieties differ from those of native human chorionic gonadotropin-ß. Endocrinology 1988;122:173-180.[Abstract]
-
Blithe DL, Wehmann RE, Nisula BC. Carbohydrate composition of ß-core. Endocrinology 1989;125:2267-2272.[Abstract]
-
Karas M, Hillenkamp F. Laser desorption ionisation of proteins with molecular masses in excess of 10000 daltons. Anal Chem 1988;60:2299-2301.[Medline]
[Order article via Infotrieve]
-
Sutton C, O’Neill JA, Cottrell JS. Site-specific characterization of glycoprotein carbohydrates by exoglycosidase digestion and laser desorption mass spectrometry. Anal Biochem 1994;218:34-36.[ISI][Medline]
[Order article via Infotrieve]
-
Lee CL, Iles RK, Shepherd JH, Hudson CN, Chard T. The purification and development of a radioimmunoassay for ß-core fragment of a human chorionic gonadotrophin in urine: application of a marker of gynaecological cancer in premenopausal and postmenopausal women. J Endocrinol 1991;130:481-489.[Abstract/Free Full Text]
-
Kussmann M, Nordhoff E, Rahbek-Nielsen H, Haebel S, Rossel-Larsen M, Jakobsen L, et al. Matrix-assisted laser desorption/ionization mass spectrometry sample preparation techniques designed for various peptide and protein analytes. J Mass Spectrom 1997;32:593-601.
-
Kardana A, Elliott MM, Gawinowicz MA, Birken S, Cole LA. The heterogeneity of human chorionic gonadotropin (hCG). I. Characterization of peptide heterogeneity in 13 individual preparations of hCG. Endocrinology 1991;129:1541-1550.[Abstract]
The following articles in journals at HighWire Press have cited this article:
|
|
|
|
 
L. Valmu, H. Alfthan, K. Hotakainen, S. Birken, and U.-H. Stenman
Site-specific glycan analysis of human chorionic gonadotropin {beta}-subunit from malignancies and pregnancy by liquid chromatography--electrospray mass spectrometry
Glycobiology,
December 1, 2006;
16(12):
1207 - 1218.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
U.-H. Stenman, A. Tiitinen, H. Alfthan, and L. Valmu
The classification, functions and clinical use of different isoforms of HCG
Hum. Reprod. Update,
November 1, 2006;
12(6):
769 - 784.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Gervais, Y.-a. Hammel, S. Pelloux, P. Lepage, G. Baer, N. Carte, O. Sorokine, J.-m. Strub, R. Koerner, E. Leize, et al.
Glycosylation of human recombinant gonadotrophins: characterization and batch-to-batch consistency
Glycobiology,
March 1, 2003;
13(3):
179 - 189.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
