Improved reagents for newborn screening of mucopolysaccharidosis types I, II, and VI by tandem mass spectrometry

Article abstract of DOI:10.1021/ac5004135

Tandem mass spectrometry for the multiplex and quantitative analysis of enzyme activities in dried blood spots on newborn screening cards has emerged as a powerful technique for early assessment of lysosomal storage diseases. Here we report the design and process-scale synthesis of substrates for the enzymes α-l-iduronidase, iduronate-2-sulfatase, and N-acetylgalactosamine-4- sulfatase that are used for newborn screening of mucopolysaccharidosis types I, II, and VI. The products contain a bisamide unit that is hypothesized to readily protonate in the gas phase, which improves detection sensitivity by tandem mass spectrometry. The products contain a benzoyl group, which provides a useful site for inexpensive deuteration, thus facilitating the preparation of internal standards for the accurate quantification of enzymatic products. Finally, the reagents are designed with ease of synthesis in mind, thus permitting scale-up preparation to support worldwide newborn screening of lysosomal storage diseases. The new reagents provide the most sensitive assay for the three lysosomal enzymes reported to date as shown by their performance in reactions using dried blood spots as the enzyme source. Also, the ratio of assay signal to that measured in the absence of blood (background) is superior to all previously reported mucopolysaccharidosis types I, II, and VI assays.

Full text of DOI:10.1021/ac5004135

Article
pubs.acs.org/ac
Open Access on 04/02/2015
Improved Reagents for Newborn Screening of
Mucopolysaccharidosis Types I, II, and VI by Tandem Mass
Spectrometry
Naveen Kumar Chennamaneni,†,∥ Arun Babu Kumar,†,∥ Mariana Barcenas,† Zdenek Spacil,†
̌
́
̌
C. Ronald Scott,‡ Frantisek Turecek,† and Michael H. Gelb*,†,§
̌
̌
†Departments of Chemistry, Pediatrics, and Biochemistry, University of Washington, Seattle, Washington 98195-1700, United
‡
§
States
S
* Supporting Information
ABSTRACT: Tandem mass spectrometry for the multiplex
and quantitative analysis of enzyme activities in dried blood
spots on newborn screening cards has emerged as a powerful
technique for early assessment of lysosomal storage diseases.
Here we report the design and process-scale synthesis of
substrates for the enzymes α-L-iduronidase, iduronate-2-
sulfatase, and N-acetylgalactosamine-4-sulfatase that are used
for newborn screening of mucopolysaccharidosis types I, II,
and VI. The products contain a bisamide unit that is
hypothesized to readily protonate in the gas phase, which
improves detection sensitivity by tandem mass spectrometry.
The products contain a benzoyl group, which provides a useful
site for inexpensive deuteration, thus facilitating the prepara-
tion of internal standards for the accurate quantification of enzymatic products. Finally, the reagents are designed with ease of
synthesis in mind, thus permitting scale-up preparation to support worldwide newborn screening of lysosomal storage diseases.
The new reagents provide the most sensitive assay for the three lysosomal enzymes reported to date as shown by their
performance in reactions using dried blood spots as the enzyme source. Also, the ratio of assay signal to that measured in the
absence of blood (background) is superior to all previously reported mucopolysaccharidosis types I, II, and VI assays.
ewborn screening of a subset of lysosomal storage
diseases is an important new area of laboratory medicine
screening for lysosomal storage diseases19,21−24 (as well as
unpublished studies in Taiwan and Illinois).
N
since treatments are available for these diseases, and early
initiation of treatment often leads to a better clinical
outcome.1,2 We have been developing the use of tandem
mass spectrometry for the multiplex assay of several lysosomal
enzymes in dried blood spots on newborn screening cards.3−8
The method allows for the quantification of enzymatically
formed product by including an internal standard, which is
structural similar to the enzymatic product or structurally
identical but with heavy isotope substitution. The methodology
has been provided in different formats for the sample
preparation steps prior to mass spectrometry.9−18 The simplest
solution that works well seems to be liquid−liquid extraction
with organic solvent followed by flow injection into the tandem
mass spectrometer.15,19
Reagents for tandem mass spectrometry analysis of six
lysosomal enzymes are currently prepared by Genzyme
Corporation and distributed by the Centers for Disease
Control and Prevention. The latter has also developed
standard, quality control dried blood spots, thus providing a
consistent set of samples to be used by newborn screening
laboratories worldwide.20 These reagents have been used in
different pilot studies, which have helped to validate newborn
Scheme 1 shows the structure of the substrates developed by
us previously for the tandem mass spectrometry assay of the
enzymes relevant to Pompe disease and mucopolysacchar-
idosis-I (MPS-I).3,5 The MPS-I substrate contains the
fluorogenic 7-hydroxycoumarin unit, thus allowing this reagent
to be used for fluorometric assays of α-L-iduronidase (the
enzyme relevant to MPS-I) in addition to tandem mass
spectrometry. However, it is now clear that tandem mass
spectrometry-based lysosomal enzyme analysis offers distinct
advantages over fluorometric assays. First, the background
signal seen in the tandem mass spectrometry assay when
substrate is omitted is virtually zero (i.e., no components of
blood other than enzymatic product are detected in the product
mass spectrometry ion-selective channels), whereas nonproduct
components of the blood contribute to significant autofluor-
escence in the fluorometric assay. This leads to a much higher
assay-to-blank ratio for the tandem mass spectrometry assay
Received: January 29, 2014
Accepted: April 2, 2014
Published: April 2, 2014
© 2014 American Chemical Society
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Scheme 1
Scheme 2
versus the fluorometric assay and to a much more accurate
assessment of enzymatic activity in the low-end range. This in
turn is expected to lead to a much lower rate of false positives
in newborn screening of lysosomal storage diseases. Fur-
thermore, there is no fluorometric assay reagent that leads to
reliable newborn screening of Niemann−Pick type-A/B disease
as available fluorometric reagents give a significant false
negative rate. This is due to a prevalent pathogenic mutation
in the population that leads to low enzymatic activity with the
natural sphingomyelin substrate but a high activity with the
artificial fluorometric substrate.25,26 The tandem mass spec-
trometry assay uses a substrate that is similar in structure to the
natural substrate (the only difference is the length of the fatty
acyl group attached to the sphingosine amino group); the false
negative problem is not an issue with this technique.
The Pompe and MPS-I products are shown in Scheme 2.
Recently, we have observed that the Pompe product gives an
∼10-fold higher signal per mole in the tandem mass
spectrometer compared to the MPS-I product.12 A detailed
theoretical study suggests that the bisamide unit of the Pompe
product allows for a particularly stable protonated ion species in
the gas phase as shown in Scheme 2.27 Given this enhanced
sensitivity and the lack of need for the fluorometric 7-
hydroxycoumarin unit, we decided to prepare new substrates
for MPS-I, -II, and -VI that contain the type of aglycone present
in the Pompe reagent. These new reagents are also simpler to
synthesize compared to the 7-hydroxycoumarin containing
reagents. In the current study we present synthesis of the new
reagents, show that they can be scaled up to amounts needed to
support worldwide newborn screening, and show that they
display enhanced tandem mass spectrometry performance
compared to the original reagents.
EXPERIMENTAL SECTION
■
Materials. Full details for the synthesis of all new reagents is
given as Supporting Information. Patent applications have been
filed with the U.S. Patent Office for the compounds described
in this study. Buffers to assay MPS-I, -II, and -VI were as
follows: MPS-I, 100 mM ammonium formate (Sigma), pH 4.4,
10 g/L sodium taurocholate (Sigma); MPS-II, MPS-I buffer
plus 7.5 mM barium(II) acetate (Sigma), 5.0 mM cerium(III)
acetate (Sigma); MPS-VI, MPS-II buffer plus 150 μM 2-
acetamido-2-deoxy-glucono-1,5-lactone (Santa Cruz Biotech-
nology). All dried blood spots were obtained with Institutional
Review Board (IRB) approval at the University of Washington.
Dried blood spots were stored at −10 to −20 °C in closed jars
with desiccant.
Enzymatic Assays. MPS-I assays used a 3 mm dried blood
spot punch in a 96-deep well plate (1 mL round-bottom
Corning Costar, Fisher, cat. no. 09-761-116A). To the punch
was added 30 μL of buffer containing 500 μM MPS-I substrate
and 3.5 μM MPS-I internal standard. Substrate and internal
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Scheme 3
Scheme 4
standard were added to a vial from stock solutions in methanol,
the solvent was removed in a centrifugal evaporator (Speed-
Vac), and assay buffer was added followed by vortex mixing to
prepare the stock solution, which could be stored at −10 to
−20 °C for at least a few months. The plate was sealed with
sealing film (AxySeal, VWR International, cat. no. 10011-117)
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for overnight (16 h) incubation at 37 °C with orbital shaking
(250 rpm).
separation resolution. These aglycones have been prepared on
the 100 g scale.
After incubation, 100 μL of a 1:1 mixture of ethyl acetate/
methanol was added to each well with a multichannel pipet to
quench the reaction. Additional 400 μL of ethyl acetate and 200
μL of water were added to each well, and mixing was achieved
by pumping up and down with the pipettor 20 times over a few
minutes. Plates were covered with polyester-based sealing film
and centrifuged at 3000 rpm for 5 min at room temperature to
separate the two liquid layers. Aliquots (200 μL) of the top
liquid phase were removed from the plate and transferred to a
new 96-well plate (0.5 mL, Axygen Scientific, VWR Interna-
tional, cat. no. 47743-982), the solvent was evaporated under a
nitrogen stream at room temperature, and samples were
reconstituted in 100 μL of the infusion solvent (80:20
methanol/water with 5 mM ammonium formate). The plates
were carefully sealed with aluminum foil and subjected to flow
injection tandem mass spectrometry on a Waters Xevo TQ
instrument. Autosampler and mass spectrometer settings are
given in Supporting Information.
MPS-II assays were carried out as above for MPS-I but with 1
mM MPS-II substrate and 5 μM MPS-II internal standard.
After incubation, 200 μL of 44 mM citric acid in water was
added to each well with a multichannel pipet to quench the
reaction and to protonate the carboxylate of the iduronic acid
residue of the product to allow extraction into organic solvent.
Ethyl acetate (400 μL) and water (100 μL) were added to each
well, and samples were mixed as above. Plates were additionally
processed as for MPS-I.
MPS-VI assays were carried out as above but with 1 mM
MPS-VI substrate and 5 μM MPS-VI internal standard. After
incubation, two different work-up protocols were used. In one
variation the reaction was quenched with 100 μL of 1:1 ethyl
acetate/methanol. Additional 400 μL of ethyl acetate and 100
μL of water were added to each well, and plates were processed
as for MPS-I assays. In the second variation, quenching of
incubated samples was with 100 μL of a suspension of DEAE
cellulose (Whatmann DE52 preswollen, 16 mg of resin per 100
μL of water) followed by 400 μL of ethyl acetate. The samples
were processed as above for the MPS-I assays.
Extraction Studies. The various enzyme products (300
pmol) were dissolved in 30 μL of assay buffer to give 10 μM
analyte. The sample was processed by liquid−liquid extraction
as above and submitted to flow injection tandem mass
spectrometry as above. The amount of product detected by
mass spectrometry was compared to that measured when 300
pmol of enzymatic product was directly injected into the
instrument. The numbers were corrected for dilution factors to
obtain the percent of total product added to assay buffer that
was recovered in the organic phase.
MPS-I substrate was made by condensing aglycone 3b with
protected iduronyl-F 45 to give 5b. Deacetylation afforded 6b,
and saponification afforded MPS-I substrate (Scheme 4).
Aglycone 3b is the MPS-I product. MPS-I internal standard
was prepared using a route similar to that used for MPS-I
product but using commercially available and inexpensive
pentadeuteriobenzoyl chloride (Supporting Information). It is
chemically identical to MPS-I product but containing five
deuteriums in the benzoyl group.
MPS-II substrate was prepared from aglycone 3c (Scheme 3)
by condensation with 4 (Scheme 4). After deacetylation, 6c was
selectively sulfated on the 2-OH group by formation of the
dibutyl stannylene acetal and treatment with SO3−Et3N as
described.30 Saponification of the methyl ester afforded MPS-II
substrate.
To prepare MPS-VI substrate, aglycone 3a was condensed
with protected N-acetylgalactosamine-1-Cl 7 followed by
deacetylation to give MPS-VI product. The latter was
selectively benzoylated on the 3- and 5-OH groups, the 4-
OH was sulfated, and debenzoylation afforded the MPS-VI
substrate (Scheme 4).
All of these new reagents contain the same bisamide unit
found in the Pompe reagent and should form the stable
protonated species shown in Scheme 2. The new reagents are
also easier to prepare than the original reagents owing to the
lack of the 7-hydroxycoumarin unit. MPS-II and MPS-VI
substrates contain longer acyl chains attached to the secondary
amine of the aglycone compared to MPS-I substrate. This is
because, prior to tandem mass spectrometry, assay mixtures are
subjected to liquid−liquid extraction with ethyl acetate to
remove buffer salts that would interfere with the electrospray
ionization process. Since MPS-II and MPS-VI products contain
a monosaccharide, we used longer acyl groups to drive the
products into ethyl acetate. We measured the fraction of MPS-I,
-II, and -VI products that are extracted into ethyl acetate by
submitting the extracts to tandem mass spectrometry analysis
compared to that of a standard solution. Extraction yields were
found to be 70%, 61%, and 30% for MPS-I, -II, and -VI
products, respectively. Incomplete extraction is tolerated since
the internal standards will be extracted to the same extent as the
corresponding products.
Tandem mass spectrometry analysis of MPS-I, -II, and -VI
products show that all three give rise to a major fragment ion
due to cleavage pathway shown in Scheme 5. Cleavage of the
precursor ions along a major reaction pathway improves
sensitivity of product detection as formation of multiple
product ions would decrease the signal intensity since only a
single ion reaction is monitored.
Enzymatic Assays Using Dried Blood Spots. The new
substrates for MPS-I, MPS-II, and MPS-VI were tested side by
side with our original reagents (see the introduction) using a 3
mm punch of a dried blood spot from a single adult as the
enzyme source. After incubation, liquid−liquid extraction with
ethyl acetate was used to extract enzymatic product and internal
standard into a buffer salt-free, organic solvent phase since high
salt interferes with the electrospray ionization process in the
mass spectrometer. Internal standards, pentadeuterated ver-
sions of the products, were used to convert the observed mass
spectrometer signals into moles of product formed. Use of a
chemically identical internal standard accounts for any losses of
product due to many factors including absorption of analytes
RESULTS AND DISCUSSION
■
Design and Synthesis of Reagents. The synthesis of the
new MPS-I substrate, MPS-II substrate, and MS-VI substrate is
shown in Schemes 3 and 4. The aglycones were synthesized by
a common pathway (Scheme 3). Commercially available
diamino alkanes were converted to monobenzoylated species
1a,b by treatment with methyl benzoate.28,29 The resulting
primary amine underwent a Michael addition with acrylamide
2, and the resulting secondary amine was acylated with the
appropriate acyl chloride to give aglycones 3a−c in a one-pot
series of reactions. Purification steps were either recrystalliza-
tion or silica chromatography without the need for high-
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Scheme 5
Table 1. Ion Counts in the Product Tandem Mass
Spectrometer Channels for the New versus Original
Products
product ion counts product ion counts
product
MPS-I product
(original reagent)
(new reagent)
82951
1240
1304
282449
7240
MPS-II product
MPS-VI product
(ethyl acetate extraction only)
10300
MPS-VI product (quench with
anion exchanger then ethyl
acetate extraction)
1263
11221
Table 2 gives the specific activities of the lysosomal enzymes
for the original and new assay reagents. These are expressed as
micromoles of product per hour per liter of blood. Internal
standard ion counts are used to convert the observed product
ion counts to moles of products. Each 3 mm dried blood spot
punch contains the components of 3.3 μL of blood. We also
carried out control assays in which a 3 mm filter paper punch
(no blood) was included, and the sample was incubated and
processed as for the complete assay. For MPS-I, the specific
activities with the original and new reagents are similar,
showing that both substrates are hydrolyzed with similar
catalytic efficiencies. In the absence of blood, the new reagent
gives a lower background signal compared to that from the
original reagent. This presumably reflects a combination of
lower amounts of new substrate extracted into ethyl acetate
compared to the original substrate and/or lower break down of
new substrate in the electrospray ionization source compared to
the original substrate. As a result the blood to no-blood assay
response is 5.6 times higher for the new versus original assay.
For MPS-II, the specific activities measured with the original
and new substrates are similar, and the blood to no-blood ratio
is 2-fold higher with the new reagent. For MSP-VI the new
reagent is a better substrate, with a specific activity that is about
8-fold higher than that with the original substrate. When the
anion exchanger is used in the reaction quench the blood to no-
blood ratio jumps from 4.9 to 97.4 when the original reagent is
replaced with the new reagent. With the new reagent, this ratio
is smaller, 16.7, when ethyl acetate extraction alone is used,
suggesting that removing more substrate from the organic
extract by trapping it on the anion-exchange resin leads to a
decrease in background signal. This in turn suggests that most
of the background comes from substrate-to-product conversion
in the electrospray ionization source.
Table 3 gives the coefficient of variation (CV, %) for three
injections of an identical solution into the mass spectrometer
(intra-assay) or for three independent assays using three
punches from the same dried blood spot (interassay) for assays
with the original and new reagents. In all cases, CVs were
<10%. These numbers are generally consistent with CVs for
other assays used in newborn screening laboratories and
illustrate the high reproducibility of these assays.
We measured the activities of the MPS-I, MPS-II, and MPS-
VI enzymes in dried blood spots from 15 random newborns
and from an affected child (diagnosis previously confirmed by
standard methods). Figures 1−3 give a graphical description of
the results, and all numerical values are provided in Supporting
Information Tables 1−3. For MPS-I, -II, and -VI, all normals
are well-separated from the affected patient. The ratio of mean
normal activity to that of the affected child is 40 for MPS-I, 32
for MPS-II, and 17 for MPS-VI. The corresponding ratios
onto surfaces, incomplete extraction into organic solvent,
consumption of products by downstream enzymes, and
differential suppression of ion formation in the electrospray
ionization source of the mass spectrometer.
Prior to tandem mass spectrometry (MS/MS), it is helpful to
remove most of the substrate in the case of sulfatases (MPS-II
and -VI) since substrate-to-product conversion occurs to a
small extent in the electrospray ionization source (due to local
heating and possibly other factors). In the case of MPS-II, the
reaction is quenched with citric acid to protonate the
carboxylate of the iduronic acid group of the MPS-II product
so that it is well-extracted into ethyl acetate. Essentially all of
the MPS-II substrate remains in the aqueous layer since it bears
a monoanionic sulfate ester that remains deprotonated (the
conjugate base of an acid whose pKa is <0). In the case of MPS-
VI, ethyl acetate extraction alone works to some extent to
separate the charge-neutral MPS-VI product from MPS-VI
substrate bearing a sulfate monoanion, but better results are
obtained if the anion exchanger DEAE-cellulose is added during
ethyl acetate extraction to tightly trap the substrate (see below).
MPS-I substrate does not significantly fragment in the
electrospray ionization source, and it is not necessary to reduce
substrate amount prior to mass spectrometry. Also since the
assay is not acidifed prior to ethyl acetate extraction, most of
MPS-I substrate remains in the aqueous phase.
Original and new reagents were used in side-by-side assays
using multiple punches form the same dried blood spot
(obtained from a healthy adult). Table 1 gives the observed ion
counts for MPS-I, MPS-II, and MPS-VI products. The signal
for the MPS-I product was 3.4-fold higher than that for an
identical assay carried out with the original MPS-I reagent (7-
hydroxycoumarin type, see the introduction). The sensitivity
improvement factor for MPS-II product is 5.8. For MPS-VI, the
assay gives a sensitivity enhancement of 7.9- and 8.9-fold,
compared to the original reagent, if reaction quenching is with
anion-exchange resin prior to liquid−liquid extraction into ethyl
acetate or ethyl acetate extraction alone, respectively. These
results support our hypothesis that enhanced protonation of
the gas-phase product ions via the bisamide unit of the new
reagents (Scheme 2) gives a larger ion count signal.
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a
Table 2. Specific Activities for Lysosomal Enzymes Using the New and Original Assay Reagents
original reagent
new reagent
spec. act.
(μmol/h/L)
spec. act. no blood
ratio of complete assay to no-blood
assay response
spec. act.
(μmol/h/L)
spec. act. no blood
ratio
blood/no blood
enzyme
(μmol/h/L)
(μmol/h/L)
MPS-I
1.57
2.88
1.41
0.03
0.076
0.28
52.3
37.9
4.91
1.21
4.40
0.004
0.06
293.5
75
MPS-II
MPS-VI
(anion exch., EtOAc)
12.17
0.125
97.4
MPS-VI (EtOAc)
1.15
0.24
4.75
13.125
0.79
16.7
a
All specific activities are not blank-corrected.
Table 3. Coefficient of Variation for the Lysosomal Assays
original reagent
new reagent
a
b
intra-assay CV (%)
interassay CV (%)
intra-assay CV (%)
interassay CV (%)
MPS-I
6.74
1.90
6.45
4.94
9.41
5.56
8.31
5.30
1.93
8.47
2.49
1.71
8.65
2.36
5.15
MPS-II
MPS-VI (anion exch., EtOAc)
MPS-VI (EtOAc)
3.28
a
b
Intra-assay CV was calculated from triplicate injections of an identical sample into the mass spectrometer. Interasssay CV is calculated from three
independent assays carried out with three different punches of the same dried blood spot.
Figure 3.
Figure 1.
CONCLUSIONS
■
The assay performance of the new reagents for MPS-I, -II, and
-VI described in this study is superior to that obtained with our
earlier reported reagents for assay of lysosomal enzymes in
dried blood spots. The new reagents are easier to synthesize
than the previous reagents and have been made on the
multigram scale appropriate to support worldwide newborn
screening. Enhanced product ion counts in the mass
spectrometer are especially important for sulfatases (MPS-II
and -VI) since these enzymes generate products containing a
monosaccharide, which generally ionize poorer in the electro-
spray source compared to products that lack a carbohydrate
group (i.e., MPS-I). The ratio of complete assay to no-blood
background assay response with the new reagents is superior to
those reported in all previously described assays of these three
lysosomal enzymes. This in turn should lead to a larger span in
assay responses when blood samples from healthy individuals
are compared to those from disease-affected newborns. These
new MPS-I, -II, and -VI reagents are currently being used in
large-scale pilot studies in newborn screening laboratories, and
parameters including positive predictive values and false
positive rates will be reported upon completion of these trials.
Figure 2.
obtained by digital microfluidic fluorescent assays are 6.5 for
MPS-I and 3.8 for MPS-II (no data for MPS-VI).31 The results
show that the MS/MS assay greatly outperforms the
fluorescence assay in terms of difference in activity between
nonaffected and lysosomal storage disease-affected individuals.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed synthetic procedures, spectroscopic data, and mass
spectrometer instrument settings. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 206-543-7142. Fax: 206-685-8665. E-mail: gelb@
chem.washington.edu.
Author Contributions
∥N.K.C. and A.B.K. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Support of this work by NIH-NIDDK (Grant R01 DK067859)
is gratefully acknowledged.
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