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Articles |
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Biochemical Genetics Laboratory, Department of Laboratory Medicine and Pathology,
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Biomedical Mass Spectrometry and Functional Proteomics Facility, Department of Biochemistry and Molecular Biology, and
3
Clinical Pharmacology Unit, Department of Pharmacology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905.
a Author for correspondence. Fax 507-266-2888; e-mail
o'brien.john{at}mayo.edu.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Serum (25 µL) was diluted with 100 µL of water before application to an immunoaffinity column that sequestered Trf isoforms. Trf isoforms were eluted from the immunoaffinity column, concentrated on a C4 column, eluted from the C4 column, and introduced into the mass spectrometer. Analysis of the Trf isoforms was entirely automated and completed in <10 min per sample.
Results: The LC-MS method demonstrated that the major abnormal Trf isoforms in CDG lack one complete oligosaccharide structure (mono-oligosaccharide) or both oligosaccharide structures (a-oligosaccharide), but not the sialic acids, as presumed on the basis of IEF methods. Calculation of relative ratios among three possible species (mono-/di-oligosaccharide and a-/di-oligosaccharide) is reproducible [mean intra- and interassay CVs were 9.3% (n = 10) and 10% (n = 5), respectively]. A reference range for patients <18 years was determined by analysis of 209 samples (for mono-/di-oligosaccharide, the median was 0.041 and the range was 0.018–0.083; for a-/di-oligosaccharide, the median was 0.007 and the range was 0.002–0.036). Comparison of data obtained with an affinity chromatography-IEF method and the LC-MS method demonstrated equivalence in the interpreted results (n = 170).
Conclusions: Advantages of the LC-MS method include improved sensitivity, minimal sample preparation, and an analysis time of <10 min. The method was automated, which allowed high throughput, with >100 samples analyzed in a single day. Moreover, the nature of the oligosaccharide defect in CDG is accurately reflected by mass resolution, and subtle oligosaccharide truncations may also be detected by this method.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) (2). Abnormal Trf isoforms, commonly referred to as
carbohydrate-deficient Trfs (CDTs), are biochemical markers for
congenital disorders of glycosylation (CDG) and chronic alcohol
consumption (3)(4). CDG are autosomal recessive
disorders presenting in infancy or early childhood with
neurodevelopmental delay (5). Dysmorphic features such as
unusual distributions of subcutaneous fat and cerebellar hypoplasia
also occur in children affected with the most common clinical
phenotype. Trf is the preferred biochemical marker for diagnostic
analysis, primarily because of its abundance in serum. However, other
glycosylated proteins, including peptide hormones, are abnormal in CDG
as well and are the likely cause of the endocrine function
abnormalities that are among the constellation of symptoms
(6). The biochemical deficiencies that cause the most
frequent forms of CDG have been identified and are caused by mutations
in the phosphomannomutase (PMM) and phosphomannose isomerase (PMI)
genes (7)(8).
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Isoelectric focusing (IEF) is the most commonly used method for detection of CDT and is considered the gold standard for the diagnosis of CDG (9). Typically, five sialic acid residues contribute to the acidic pI of Trf, and loss of these residues causes a basic shift within the pI of 5.2–5.9. Current methods for specific identification of Trf isoforms use combinations of IEF in conjunction with Western blotting or immunopurification-IEF followed by Coomassie staining (4). Demonstration of CDT for CDG diagnosis by either of the above sequences is adequately done by IEF. More recently, microcolumn separation followed by a turbidimetric immunoassay or HPLC has been introduced to report CDT as a relative amount compared with total serum Trf (10). Ion exchange in a HPLC configuration has also been demonstrated (11). As with IEF, resolution of Trf isoforms by ion-exchange chromatography is based on charge and is not affected by neutral monosaccharide moieties. This has led to the conclusion that CDT is a Trf lacking sialic acid residues. This structure is not consistent with the molecular weight measurements we attribute to CDT in CDG using mass spectrometry.
Given the increasing clinical interest in CDG and the gradual transition of this procedure to a high-volume test, replacing the affinity chromatography-IEF method is highly desirable in reducing turnaround time. We have, therefore, developed a fully automated online immunoaffinity liquid chromatography–mass spectrometry (LC-MS) method for routine detection of Trf isoforms.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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immunoaffinity column preparation
Buffers and solutions from PerSeptive Biosystems were prepared
according to directions provided by the manufacturer. Rabbit anti-human
Trf (14 mg) was dialyzed overnight against 1 L of phosphate-buffered
saline (PBS; 0.01 mol/L phosphate-0.150 mol/L NaCl, pH 7.4) in
a Slide-A-Lyzer dialysis cassette. After overnight dialysis, rabbit
anti-human Trf was added to 4 mL of PBS, slowly mixed with 2.5 mL of
POROS-aldehyde high-sulfate buffer, and then mixed with sodium
cyanoborohydride to a final concentration of 5 g/L.
POROS-aldehyde self pack medium was added (150 mg), and the entire
solution was rocked gently for 1 min. POROS-aldehyde high-sulfate
buffer was added (1 mL), and the solution was agitated for another 5
min. Additional 1-mL increments of POROS-aldehyde high-sulfate buffer
were added every 5 min for a total of 5 mL. The solution was rocked for
90 min at room temperature. The immobilized ligand was filtered using a
10–20 µm sintered glass funnel, resuspended in 10 mL of
POROS-aldehyde capping buffer, and rocked for 30 min at room
temperature. The immobilized ligand was then washed using 50 mL of
POROS-aldehyde loading buffer, 50 mL of 1 mol/L NaCl, and 50 mL of
POROS-aldehyde loading buffer before being stored in 1 mL of
POROS-aldehyde loading buffer. Five C.128 columns (20 x 1 mm)
were packed at a flow rate of 2 mL/min with the immobilized ligand (17
µL/column) using POROS-aldehyde loading buffer. Columns stored for
later use were washed with 0.2 g/L sodium azide in loading
buffer.
sample preparation
A 25-µL serum sample was mixed with 100 µL of reverse-osmosis
H2O (1:5 dilution by volume). After
vortex-mixing, a 100-µL portion of the dilution was transferred to an
autosampler vial for analysis.
methods
LC-MS method.
Method development and validation were
performed using an API 3000 triple quadrupole mass spectrometer
(Perkin-Elmer Sciex) equipped with the TurboIonSpray ionization probe
source (operated at 5500 V). Peripherals included two Perkin-Elmer
Series 200 Micropumps, a Perkin-Elmer Series 200 Autosampler, and a
Shimadzu System Controller (SCL-10Avp), which controlled two Shimadzu
Liquid Chromatography LC-10ADvp pumps and two Valco two-position
actuator control modules.
A 5-µL aliquot of the diluted serum was applied to the immunoaffinity
column (20 x 1 mm column containing anti-human Trf coupled to
POROS-aldehyde medium) in PBS (pH 7.4) at a flow rate of 100 µL/min
for 2 min. All other serum components were diverted to waste because of
the configuration of valve 1 (position A). At 2 min into the run, valve
1 was switched to position B, and Trf isoforms were subsequently eluted
from the immunoaffinity column for 2 min with 100 mmol/L glycine
containing 20 mL/L acetic acid buffer (pH 2.5; flow rate, 100 µL/min)
and concentrated on a C4 cartridge [Widepore
C4 (butyl); 4 x 2.0 mm (i.d.);
Phenomenex]. The C4 column was then washed with
10 mL/L acetic acid-methanol-acetonitrile (98:1:1 by volume) at
a flow rate of 100 µL/min for 2 min to remove excess phosphate and
other buffer components that might suppress MS response. Valve 2 was
switched to position B 6 min into the run, and Trf isoforms were eluted
from the C4 column and introduced into the
TurboIonSpray source using 5 mL/L acetic acid in 0.2 g/L
trifluoroacetic acid-methanol-acetonitrile (5:48:48 by volume)
at a flow rate of 100 µL/min (Fig. 2
). The TurboIonSpray source was operated with turbo gas on (6
L/min; sensor temperature, 150 °C) with the effluent flow splitting
at 1:2. The mass spectrometer was operated in Q1 scan mode
(m/z 2000 to m/z 3000) with a Trf retention time
of 
7 min and total instrument acquisition time of 9.5 min/sample.
After completion of development, the method was also validated on an
API 150 single quadrupole LC-MS system (Perkin-Elmer Sciex) equipped
with the same TurboIonSpray ionization probe source and peripherals.
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Data were acquired and processed using MassChrom software (Ver. 1.1.2; Perkin-Elmer Sciex), including BioMultiView (Ver. 1.3.1; Perkin-Elmer Sciex). The BioSpecTM-Reconstruct algorithm was used to deconvolute the charge distribution raw data to reconstructed mass data. Specifically, multiply charged spectra were transformed through five iterations, using an input data range of m/z 2000–3000 and an output data range of 74 000 to 81 000 Da. Relative quantification of CDT was achieved by comparing a-oligosaccharide Trf and mono-oligosaccharide Trf with di-oligosaccharide Trf.
IEF method.
For method comparison, affinity chromatography-IEF
was adapted from previously published procedures (4). Rabbit
anti-human Trf coupled to Sepharose was prepared by mixing rabbit
anti-human Trf with CNBr-activated Sepharose 4B (Pharmacia Amersham
Biotech) under standard coupling conditions (12). The
antihuman Trf was stored suspended in 0.1 mol/L citrate-0.025
mol/L phosphate, pH 7.2 (1:1 by volume).
Trf was isolated from 1 mL of serum by mixing it with 2 mL of
antibody-Sepharose suspension for 2 h at room temperature. After
binding, the resin and liquid were transferred to a 4-mL syringe
column fitted with a frit (Alltech). The binding mixture was expelled,
and unbound proteins were eluted with eight 2-mL washes of the binding
buffer (0.01 mol/L citrate-0.025 mol/L phosphate, pH 7.2). The Trf was
eluted with four 1-mL washes of elution buffer (0.1 mol/L citrate-0.025
mol/L phosphate, pH 2.9), and immediately neutralized with
Na2HPO4 to a pH of 7.2.
After overnight dialysis of the neutralized eluate vs 4 L of 0.5 mol/L
Na2HPO4, the Trf was iron
saturated by incubation with 50 mL of 20 mmol/L ferric citrate for
1 h at 37 °C. The Trf was concentrated in Amicon Centricon
YM-30 (Millipore) microconcentrators, and protein was determined by the
Lowry method (13). Serum Trf preparations were diluted to
0.5–1.0 g/L protein depending on their concentration, and 15
µL was applied to the focusing gels. The IEF gels consisted of 6%
acrylamide, pH 3.0–10.0. The gels were focused until 1940 V-h were
reached at 5 W (2 h). The gels were fixed with a mixture of 115 g/L
trichloroacetic acid and 35 g/L 5-sulfosalicylic acid for 30 min,
rinsed with reverse-osmosis H2O, and then soaked
in reverse-osmosis H2O for 1 h. The Trf
bands were visualized by staining with 5 g/L Coomassie Brilliant Blue
in 250 mL/L ethanol and 100 mL/L acetic acid and destaining for 1
h with 400 mL/L methanol and 100 mL/L acetic acid. The gels were
preserved for photography and densitometry by soaking in 25 mL/L
glycerol for 15 min. Scanning was accomplished using the Helena Rep
densitometer (Helena Laboratories). Results were considered abnormal if
the asialo and monosialo bands were >5% and >6%, respectively,
and/or the disialo band was >15% of the total Trf as determined by
the densitometer (4). The total time to setup and process a
batch of 24 samples was 3 days, including preparation of acrylamide
gels 24–48 h before use.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, with Trf eluted at 7 min in a 9-min analysis. The
BioSpec-Reconstruct algorithm deconvolutes charge distribution raw data
(Fig. 3B
) to reconstructed mass data with normal control serum showing
a single ion at 79 561 Da (Fig. 3C
), a value in close agreement to the
calculated mass of di-oligosaccharide Trf (79 555.415 Da) as reported
in the Swiss Protein Data Base. Trf immunopurified from serum of
patients diagnosed with CDG (both PMM and PMI) revealed three distinct
species when analyzed by LC-MS (Fig. 4
). The three ions at 79 561, 77 353, and 75 145 Da
present in CDG serum correspond to the intact glycoprotein, loss of one
oligosaccharide moiety (loss of 2208 Da), and loss of two
oligosaccharide moieties (loss of 4416 Da), respectively. These
isoforms correspond to those called disialo and asialo bands,
respectively, when resolved by IEF. The within- and between-run
stability of the reconstructed masses are summarized in Table 1
.
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precision and stability
Precision data were determined by replicate analyses of one normal
and three abnormal serum specimens on a single day (n = 10),
followed by a single analysis on subsequent days (n = 5), and are
summarized in Table 2
(mean intra- and interassay CVs were 9.3% and 10%,
respectively). The data show that inter- and intraassay CVs
were reproducible over control and abnormal oligosaccharide ratio
ranges.
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The stability of Trf isoforms after initial serum dilution was
determined by repeat injections of four samples covering control and
abnormal oligosaccharide ratio ranges. Samples were stored at room
temperature between injections. Table 3
shows data obtained and indicates stability of Trf isoforms up
to 96 h after initial dilution.
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reference range determination
The reference range for pediatric patients (median age, 3 years;
range, 1 day to 17 years) was determined using 209 control sera (for
the mono-/di-oligosaccharide ratio, the median was 0.041 and the range
was 0.018–0.083; for the a-/di-oligosaccharide ratio, the median was
0.007 and the range was 0.002–0.036). The distribution of results is
shown in Fig. 5
and compared with the 24 CDG cases described below.
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method comparison
Unused portions of 170 serum samples routinely analyzed by
affinity chromatography-IEF were reanalyzed by LC-MS. Of these samples,
24 were from patients for whom either the enzyme deficiency (PMM or
PMI) was detected (n = 12) or the original abnormal IEF pattern
was confirmed by an independent laboratory (n = 12). Result
interpretation based on the isoform ratio data indicated equivalence
for both control and CDG specimens vs the IEF method (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In conclusion, LC-MS allows the detection of Trf isoforms in as little as 5 µL of serum with an analysis time <10 min/sample, two major improvements over IEF (required volume, 1 mL) or turbidimetric immunoassay (100 µL), which also paves the way to a possible application of this method to the analysis of dried blood spots. This method does not require iron loading, which is an important advantage because it is well documented that the loss of one iron residue from Trf causes an artifactual pI shift nearly equivalent to the loss of a sialic acid residue (9), mimicking a CDG-like pattern. LC-MS offers numerous technical advantages over our current affinity chromatography-IEF method and other methods. It is rapid, requires virtually no sample preparation, and the online steps have been completely automated. The use of acrylamide and IEF for routine clinical tests is undesirable for both safety and convenience reasons. Although the affinity chromatography-IEF method used previously in our laboratory requires 3–4 days for completion, much of the time is not "hands-on", and shortening of the process could be achieved; however, it never approaches an output of >100 samples per day. In our estimates, personnel effort and total cost (supplies and equipment depreciation cost, based on the less expensive single-stage LC-MS system) were reduced by 75% and 93%, respectively.
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Acknowledgments |
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Footnotes |
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References |
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