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Articles |
ek Ture
ek1a
Departments of
1
Chemistry,
2
Pediatrics, and
3
Biochemistry,
University of Washington, Box 351700, Seattle, WA 98195-1700.
aAddress correspondence to this author at: Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195-1700. Fax 206-685-3478; e-mail turecek{at}chem.washington.edu b address correspondence to this author at: Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195-1700. Fax 206-685-8665; e-mail gelb{at}chem.washington.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Methods: We developed a sensitive and specific method to assay ASM and GCG in skin fibroblast homogenates using biotinylated substrate conjugates. The products were purified by bioaffinity capture on streptavidin-agarose beads and, following release, were analyzed by electrospray ionization mass spectrometry. Quantification was achieved using stable-isotope-labeled internal standards that were chemically identical to the products of the enzymatic reactions.
Results: The method demonstrated excellent linearity of ASM and GCG enzymatic product formation with the amount of cellular protein and incubation time. The range of ASM activities in fibroblast lysates from six healthy patients was 39–70 nmol · mg-1 · h-1 compared with 3.7–5.1 nmol · mg-1 · h-1 in cell lysates from two patients affected with Niemann-Pick A disease. The GCG activities toward the corresponding substrate conjugate were 4.0–6.8 nmol · mg-1 · h-1 in cell lysates from healthy patients compared with 0.1–0.2 nmol · mg-1 · h-1 in cell lysates from two patients affected with Krabbe disease. The amounts of substrate conjugates needed per analysis were 15 nmol (14 µg) for both ASM and GCG.
Conclusions: Electrospray mass spectrometry combined with the use of biotinylated substrate conjugates and bioaffinity purification represents a new approach for the diagnosis of lysosomal storage diseases as demonstrated for Niemann-Pick A and Krabbe diseases. No radioactive substrates are used, and the method uses a single instrumental platform to determine both ASM and GCG in one cell sample.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Our general strategy for enzyme assays using mass spectrometry is shown
in scheme 1
(12)(13). Action of the enzyme on the
substrate conjugate causes chemical modification in the substrate
moiety or conjugate cleavage. These chemical changes are accompanied by
a change in the molecular mass of the product, which is detected by
mass spectrometry following electrospray ionization.
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A key feature for rapid analysis is the presence in the substrate conjugate, and thus in the product, of a biotin moiety (molecular handle) that allows specific capture by streptavidin immobilized on agarose beads; nonbiotinylated species can then be removed by simple washing. This approach avoids tedious purification by chromatography and has the potential for automation. Another key feature pertinent to quantification by mass spectrometry is the use of internal standards labeled with stable isotopes (2H, 13C, or a combination thereof). The internal standards are chemically identical to the products of the enzymatic reactions but differ in molecular mass as a result of the presence of heavy isotopes. The internal standards are added at the end of the enzymatic reaction. After affinity capture of biotinylated conjugates and clean-up by multiple washings, the biotinylated conjugates are displaced from the beads with excess free biotin, and the final wash solution is directly analyzed by ESI-MS.
The advantages of using ESI-MS are several-fold. One advantage is that ESI is a gentle ionization method that typically does not lead to fragmentation of gas-phase ions (11). The information obtained from ESI-MS therefore relates to the intact molecules of the analyte that appear as cations with m/z values corresponding to that of the analyte plus that of the charge-introducing particle, which typically is a proton or a sodium cation. Analysis in the negative-ion mode is also possible if dictated by the chemical nature of the conjugate. Another advantage is that ESI-MS as a rule provides excellent detection limits, often in the low femtomole (10-15 mol) to attomole (10-18 mol) range. Finally, the m/z values are highly indicative of the analyte species, in particular when coupled with an isotopically labeled derivative of known mass difference.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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85%), psychosine, Triton X-100,
sodium taurocholate, and oleic acid were purchased from Sigma. Human
skin fibroblasts from two patients with Niemann-Pick disease and two
with Krabbe disease, repository numbers GM00406, GM00370, GM04517, and
GM04372, were obtained from the NIGMS Human Genetic Mutant Cell
Repository (Camden, NJ). These cells as well as fibroblasts from six
healthy persons were cultured according to standard procedures
(14) and stored at -80 °C.
substrate conjugates and internal standards
The substrate conjugates for ASM and GCG consisted of similar
building blocks (schemes 2
and 3
). Biotin was conjugated to a sarcosine
linker that served two functions: (a) it provided the moiety
for stable-isotope introduction in the synthesis of internal standards,
and (b) it blocked the action of the enzyme biotinidase
(15), which is present in biological fluids and could cleave
the biotin handle off the product conjugates. Sarcosine was further
conjugated to 12-aminododecanoic acid (for ASM) or
11-aminoundecanoic acid (for GCG) linkers that mimicked the
hydrophobic chain in the natural sphingomyelin or cerebroside
substrates (7). The different linker chain lengths for ASM
and GCG were chosen to offset the molecular masses of the products and
internal standards and to allow simultaneous assay of both enzymes in
one sample. The carboxylic acid groups provided the functionalities for
coupling to the amino group in the sphingosine moiety. The chemical
coupling of the handle, linker, and substrate parts was achieved
through stable amide bonds to ensure chemical and enzymatic stability
of the substrate conjugates and products. The chemical syntheses of the
substrate conjugates and internal standards labeled with stable
isotopes are described below.
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ASM cleaved the choline moiety and reduced the molecular mass of the
substrate conjugate from 958.6 Da to that of the product (793.6 Da).
This change was readily detected by ESI-MS as the corresponding (M +
H)+ ions at m/z 959.7 and 794.6,
respectively (scheme 2
). The internal standard was shifted by 4 mass units as a
result of the presence of the 2H and
13C labels in the sarcosine linker. The stable
isotopes were introduced in positions that do not undergo exchange with
solvent or reagents, ensuring chemical homogeneity of the internal
standard. Likewise, GCG cleaved galactose and reduced the molecular
mass of the substrate conjugate from 941.6 Da to that of the product
(779.6 Da). The reaction progress was monitored by the appearance of
the (M + H)+ ion of the product at m/z
780.6 and quantified by the peak of the internal standard at
m/z 784.6 (scheme 3
).
synthetic procedures
Biotin tetrafluorophenyl ester
(1).
Biotin (2.0 g; 8.18 mmol) was dissolved in 40 mL of warm (60 °C)
N,N-dimethylformamide (DMF) under argon
atmosphere. After the solution cooled to room temperature, 2 mL (14.3
mmol) of triethylamine was added, followed by 3.2 g (12.2 mmol) of
2,3,5,6-tetrafluorophenyl trifluoroacetate (12). The
reaction was stirred at room temperature for 30 min, and the solvent
was removed under reduced pressure. The product was triturated
with 100 mL of ether and filtered. The isolated product was dried under
reduced pressure to yield 2.4 g (75%) of biotin tetrafluorophenyl
ester 1 as a white solid. Proton nuclear magnetic resonance
(1H-NMR; 500 MHz, CD3OD)
gave the following results: 8.02 (s, 1H), 5.62 (s, N-H), 5.16 (s, N-H),
4.54 (m, 1H), 4.34 (m, 1H), 3.17 (m, 1H), 3.02–2.08 (m, 2H),
2.65–2.80 (t, 2H), 1.87–1.55 (m, 4H), 1.40 (m, 2H), where s indicates
a singlet, d indicates a doublet, t indicates a triplet, and m
indicates a multiplet.
N-Methyl-N-biotinylglycine methyl ester
(2).
Biotin tetrafluorophenyl ester (1.00 g; 2.55 mmol) in 10 mL of
anhydrous DMF under argon atmosphere was added to a mixture of 0.482 g
(3.45 mmol) of N-methylglycine methyl ester hydrochloride
dissolved in 5 mL of anhydrous DMF and 0.5 mL (3.59 mmol) of
triethylamine. The reaction mixture was stirred at room temperature for
2 h, and the solvent was removed under reduced pressure. The
residue was extracted with chloroform (twice with 40 mL each time),
washed with water (twice with 10 mL each time), and dried with
anhydrous sodium sulfate. The solvent was removed under reduced
pressure to yield 0.801 g (95%) of compound 2.
1H-NMR (500 MHz, CD3OD)
gave the following results: 6.36 (s, N-H), 5.82 (s, N-H), 4.49 (m, 1H),
4.32 (m, 1H), 4.07–4.17 (m, 2H), 3.71 (s, 3H), 3.17 (m, 1H), 3.08 and
2.96 (s + s, 3H), 2.97–2.70 (m, 2H), 2.42 and 2.27 (t + t, 2H),
1.82–1.60 (m, 4H), 1.42 (m, 2H). ESI-MS gave a peak at m/z
330.4 (M + H)+.
N-Methyl-N-biotinylglycine
(3). Ester
2 (0.801 g; 2.43 mmol) was hydrolyzed in a mixture
containing 15 mL of methanol and 10 mL of 2 mol/L NaOH at room
temperature with stirring for 2 h. The mixture was diluted with 20
mL of methanol–H2O (50:50 by volume) and
neutralized with a cation-exchange resin (AG MP-50 Resin,
H+ form). The solution was filtered, and the
resin was washed with methanol–H2O (50:50 by
volume). The solutions were combined, and the combined solvent was
removed under reduced pressure to yield 0.620 g (80.8%) of compound
3. 1H-NMR (500 MHz,
CD3OD) gave the following results: 4.56 (m, 1H),
4.38 (m, 1H), 4.19–4.04 (m, 2H), 3.27 (m, 1H), 3.20 and 3.04 (s + s,
3H), 2.98–2.75 (m, 2H), 2.50 and 2.33 (t + t, 2H) 1.60–1.87 (m, 4H),
1.44–1.59 (m, 2H). ESI-MS gave a peak at m/z 316.4 (M +
H)+.
N-Methyl-N-biotinylglycine tetrafluorophenyl ester
(4). Compound 3 (95.6 mg; 0.3 mmol) was
dried under reduced pressure overnight. Dry DMF (4 mL) was added, and
the mixture was stirred with mild heating for 10 min. Freshly distilled
triethylamine (100.3 µL) was then added, followed after 3 min by 32
µL of tetrafluorophenyl trifluoroacetate. Another 32 µL of
tetrafluorophenyl trifluoroacetate was added after a 5-min interval.
The mixture was stirred for 1 h under argon. Thin-layer
chromatographic analysis showed formation of the
tetrafluorophenyl ester 4
(Rf 0.5; mobile phase,
CHCl3–methanol, 9:1 by volume).
N-(N'-Methyl-N'-biotinylglycyl)12-aminododecanoic
acid
(5a). To the solution of
4 in DMF was added 77.5 mg (0.36 mmol) of 12-aminododecanoic
acid in 4 mL of DMF, and the mixture was stirred at room temperature
for 2 h. Chloroform (100 mL) was added, and the solution was
washed with 20 mL of 1 mol/L HCl and brine and then dried with sodium
sulfate. The solvent was removed, and the residue was redissolved in
methanol and purified by HPLC (protein & peptide
C18 column; cat. no. 218TP1022; VYDAC ), using
the following gradient in which H2O was solvent A
and methanol was solvent B: 0–10 min, 0% B; 10–20 min, 0–30% B;
20–100 min, 30–100% B. The product was eluted at 65.4 min to yield
92.4 mg (60.1%) of compound 5a.
1H-NMR (500 MHz, CD3OD)
gave the following results: 4.54 (m, 1H), 4.37 (m, 1H), 4.10–4.00 (m,
2H), 3.29–3.20 (m, 2H), 3.11 and 2.95 (s + s, 3H), 2.95 and 2.73 (d +
d, 2H), 2.56–2.34 (m, 2H), 2.29 (t, 2H), 1.84–1.60 (m, 6H),
1.58–1.42 (m, 4H), 1.40–1.28 (m, 14H). ESI-MS gave a peak at
m/z 513.4 (M + H)+.
N-(N'-Methyl-N'-biotinylglycyl)12-aminoundecanoic acid (5b) was prepared using a method analogous to that for compound 5a.
N-Hydroxysuccinimide ester of 5a
(6a). Compound 5a (9.8 mg; 1.9
µmol) and N-hydroxysuccinimide (2.2 mg; 1.9 µmol) were
dissolved in 100 µL of anhydrous DMF, and dicyclohexylcarbodiimide
(3.9 mg; 1.9 µmol) was added last. The reaction was allowed to
proceed for 60 h at room temperature in the dark with continuous
stirring. The crude product was purified by flash chromatography on
silica gel (chloroform–methanol, 15:1–12:1 by volume) to yield 9.8 mg
(84%) of compound 6a. 1H-NMR gave the
following results: 4.52 (m, 1H), 4.26 (m, 1H), 3.10 and 2.95 (s + s,
3H), 3.00 (s, 4H), 2.90–2.73 (m, 2H), 2.56–2.42 (m, 2H), 2.38 (t,
2H), 1.82–1.60 (m, 6H), 1.57–1.42 (m, 4H), 1.40–1.26 (m, 14H).
The N-hydroxysuccinimide ester of 5b (compound 6b) was prepared using a method analogous to that for compound 6a.
ASM substrate conjugate
(7).
Sphingosylphosphoryl choline (2.5 mg; 5.4 µmol) and 6a
(3.3 mg; 5.4 µmol) were dissolved in 150 µL of anhydrous DMF, and
2.5 µL of diisopropylethylamine was added. The reaction was
allowed to proceed for 2 days at room temperature. The product was
purified by HPLC (protein & peptide C18 column;
cat. no. 218TP1010; VYDAC), using the following gradient in which
H2O was solvent A and methanol was solvent B:
0–10 min, 0% B; 10–11 min, 0–20% B; 11–60 min, 20–100% B;
60–90 min, 100% B. The product was eluted at 75.5 min to yield 3.6 mg
(69%) of compound 7. 1H-NMR (500 MHz,
CD3OD) gave the following results: 5.90–5.75 (m,
1H), 5.60–5.45 (dd, 1H), 4.64–4.50 (m, 1H), 4.47–4.35 (m, 2H),
4.26–3.98 (m, 6H), 3.82–3.71 (m, 2H), 3.45–3.3 5 (m, 2H), 3.25 (s,
9H), 3.15, 3.04 (s + s, 3H), 3.08–3.00 (m, 1H), 3.00–2.80 (m, 2H),
2.60–2.40 (m, 2H), 2.32 (t, 2H), 2.10 (m, 2H), 1.82–1.60 (m, 6H),
1.57–1.42 (m, 4H), 1.38–1.26 (m, 36H), 1.00 (t, 3H). ESI-MS gave a
peak at m/z 959.7 (M + H)+.
GCG substrate conjugate
(8). Psychosine (4.7
mg; 10 µmol) and 6b (6.0 mg; 10 µmol) were dissolved in
200 µL of anhydrous DMF, and 5 µL of diisopropylethylamine was
added. The reaction was allowed to proceed for 60 h at room
temperature. The product was purified by HPLC (protein & peptide
C18; cat. no. 218TP1010; VYDAC), using the
following gradient in which H2O was solvent A and
methanol was solvent B: 0–10 min, 0% B; 10–11 min, 0–20% B; 11–60
min, 20–100% B; 60–90 min, 100% B. The product eluted at 80.1 min
to yield 6.0 mg (64%) of compound 8.
1H-NMR (500 MHz, CD3OD)
gave the following results: 5.90–5.75 (m, 1H), 5.60–5.45 (dd, 1H),
4.60–4.50 (m, 1H), 4.25–3.90 (m, 7H), 3.85–3.75 (m, 2H), 3.65 (d,
1H), 3.55 (m, 2H), 3.50 (m, 1H), 3.29–3.20 (m, 2H), 3.11, 2.95 (s + s,
3H), 2.95 and 2.73 (d + d, 2H), 2.56–2.34 (m, 2H), 2.28 (t, 2H), 2.10
(m, 2H), 1.84–1.60 (m, 6H), 1.58–1.42 (m, 6H), 1.40–1.28 (m, 34H).
N-(p-Toluenesulfonyl)glycine-1-13C
(9). Glycine-1-13C (2.28 g;
30 mmol) was dissolved in 25 mL of 3 mol/L sodium hydroxide, and
p-toluenesulfonyl chloride (6.29 g; 33 mmol) was added. The
mixture was stirred at room temperature for 4 h. The solution was
then acidified to pH 1 with 6 mol/L hydrochloric acid. The
crystals were collected, dried, and flash-chromatographed on a
silica gel column, using ethyl acetate–CHCl3
(3:2 by volume) containing 20 mL/L acetic acid as the mobile
phase, to yield 4.42 g (64%) of compound 9.
1H-NMR (500 MHz, CD3OD)
gave the following results: 7.78–7.68 (d, 2H), 7.38–7.29 (d, 2H),
3.70 (s, 2H) 2.50 (s, 3H).
N-(Methyl-d3)-N-(p-toluenesulfonyl)glycine-1-13C
(10). Compound 9 (4.37 g; 19 mmol) in
18 mL of 4 mol/L sodium hydroxide was cooled to 0 °C. Deuterated
iodomethane (CD3I) was added, and the
solution was maintained at 75 °C for 4 h under argon. The
mixture was cooled, washed with chloroform (twice with 10 mL each), and
acidified to pH 1 with 6 mol/L hydrochloric acid. The crystals were
collected and flash-chromatographed on a silica gel column,
using ethyl acetate–CHCl3 (3:1 by volume)
containing 20 mL/L acetic acid as the mobile phase, to yield 4.70
g (82.3%) of compound 10. 1H-NMR (500
MHz, CD3OD) gave the following results:
7.76–7.68 (d, 2H), 7.38–7.29 (d, 2H), 3.96–3.90 (d, 2H), 2.50 (s,
3H).
N-(Methyl-d3)glycine-1-13C
(11). A solution of compound 10 (2.97
g; 12 mmol) in 30 mL of hydrogen bromide-saturated glacial acetic acid
was heated at 80 °C under argon for 3 h. The mixture was poured
into 120 mL of water and washed with ether; the aqueous solution was
then evaporated under reduced pressure to dryness. The solid residue
was recrystallized from acetone to give 1.88 g (90%) of compound
11. 1H-NMR (500 MHz,
CD3OD) gave the following results: 3.96–3.90 (d,
2H).
N-(Methyl-d3)glycine-1-13C methyl ester
(12). To an ice-cold solution of compound
11 (1.39 g; 8 mmol) in 15 mL of dry methanol was added
dropwise 0.3 mL of SOCl2. The solution was then
refluxed for 4 h. The solvent was evaporated under reduced
pressure, and traces of SOCl2 were removed by
adding benzene (10 mL) and repeating the evaporation three times.
Without further purification, the product was used for the following
step with an estimated yield of 90%.
ASM and GCG product conjugate internal standards
(13 and 14). The desired compounds were
made using the same procedure to make compounds 7 and
8 except that sphingosine was used in place of
sphingosylphosphoryl choline and psychosine, respectively. The
structures of compounds 13 and 14 were confirmed
by 1H-NMR and ESI-MS.
asm assay
To prepare the cell homogenate, fibroblasts were thawed, suspended
in 100 µL of purified water (MilliQ; Millipore Inc.) in a 1.7-mL
polypropylene microcentrifuge tube and lysed by two 15-s bursts of
pulsed (20% cycle) ultrasound (Model W-225 sonicator; Ultrasonics,
Inc.; output control setting 2, stainless steel probe) while cooling
the tube in an ice bath. The number of fibroblasts used was sufficient
to give a protein concentration of 2–5 g/L of lysate when
assayed using the Bradford assay (Bio-Rad) calibrated with bovine serum
albumin. The lysate was kept on ice and used within 15 min.
A mixture of 15 nmol (14 µg) of ASM substrate conjugate 7 and 75 µg of Triton X-100 in methanol was placed in a 1.7-mL polypropylene microcentrifuge tube, and the solvent was evaporated under a stream of nitrogen. After drying, 75 µL of 1.0 mol/L acetate buffer (pH 5.0) was added, and the tube was placed in a bath sonicator (Model G112SP1T; Lab Supplies Inc.) for 1.5 min. Fibroblast lysate containing 30–120 µg of protein was added, and the total volume was adjusted to 0.15 mL with MilliQ water. The sample was incubated at 37 °C for 1–4 h.
After incubation, the reaction was quenched by adding methanol (0.45
mL), and 1–4 nmol (0.7–3 µg) of internal standard 13 was
added as a 0.5 mmol/L solution in methanol. The solvent was
removed under reduced pressure (Speed-Vac; Savant Instruments), the
residue was redissolved in methanol (0.15 mL), and insoluble debris was
removed by centrifugation (12 000g for 1 min). The
supernatant (100 µL) was transferred to a Microbiospin column
(Bio-Rad) containing immobilized streptavidin (Immunopure; Pierce; 5
nmol of biotin binding capacity; suspension supplied by manufacturer).
The column was gently rocked at room temperature for 2 min and then
centrifuged (6000g for 1 min). The gel was washed by
rocking for 2 min with methanol–water (1:1 by volume; 150 µL) and
centrifuged as above. This wash step was repeated once followed by
three washes with MilliQ water (150 µL). To release the conjugates,
80 µL of a 0.5 mmol/L solution of biotin ethyl ester in methanol was
added, and the spin column was capped and incubated at 4 °C for
6 h. The column was centrifuged as above, and the eluate was mixed
with 20 µL of 20 mmol/L ammonium acetate in methanol to facilitate
ESI-MS.
gcg assay
A mixture of GCG substrate (14 µg; 15 nmol), sodium taurocholate
(100 µg; 186 nmol), and oleic acid (20 µg; 70 nmol) in
chloroform–methanol (2:1 by volume) was placed in a 1.7-mL
polypropylene microcentrifuge tube, and the solvent was evaporated
under a stream of nitrogen. After drying, 75 µL of a 0.2 mol/L
citrate-phosphate buffer (pH 4.2) was added, and the tube was sonicated
for 2 min as for the ASM assay. Typically, 20–100 µg of fibroblast
protein (cell homogenate prepared as for the ASM assay) was used per
incubation. The total volume was adjusted to 0.15 mL with MilliQ water,
and the sample was incubated at 37 °C for 1–4 h. Reactions were
worked up exactly as described for the ASM assay using 1–4 nmol
(0.7–3 µg) of the GCG internal standard 14.
esi-ms analysis
Mass spectra were obtained on a Bruker-Esquire ion trap mass
spectrometer. Samples were infused by flow injection at 1 µL/min and
ionized in a standard orthogonal Bruker ionizer. The mass spectrometric
conditions were as follows: electrospray capillary, 4000 V; end-plate,
500 V; transfer capillary, 70 V; drying gas temperature, 250 °C;
skimmer 1, 20 V; skimmer 2, 6.0 V; octopole I, 3 V; octopole II, 1 V;
octopole radiofrequency, 100 V; peak-to-peak lens I voltage,
-5 V; lens II voltage, -50 V. Mass spectra were obtained by ejecting
trapped ions in the range of m/z 700-1000 for ASM assays and
m/z 750–850 for GCG assays. Approximately 100 scans were
accumulated and averaged to provide the spectra used for
quantification. The relevant regions in the ESI mass spectra are shown
in Figs. 1
and 2
and discussed below.
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Results and Discussion |
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, bottom panel). The peaks at
m/z 959.7–961.7 correspond to the unreacted substrate
conjugate, whereas the peaks at m/z 798.8–800.8 are from
the heavy isotope-labeled internal standard. The peaks at
m/z 816.8 and 820.8 in Fig. 1
are from nonlabeled and heavy
isotope-labeled product conjugates, respectively, ionized by attachment
of a sodium ion. Both the (M + H)+ and (M +
Na)+ peaks can be used for quantification; in the
present analyses, the more abundant (M + H)+
peaks were measured. Likewise, the GCG activity was determined from the
ESI mass spectrum (Fig. 2
, bottom panel), which showed the peaks of the
enzymatic product at m/z 780.9–782.9 and those of the
labeled internal standard at m/z 785.0–787.0. The ESI mass
spectra demonstrate that the mass shift of 4 mass units in the internal
standards attributable to stable-isotope labeling was sufficient to
prevent overlap with the peaks of the enzymatic products.
Quantification of the data obtained from the ESI mass spectra provided
activities in
nmol · mg-1 · h-1
for both ASM and GCG as shown in Table 1
. The measurements showed 20% variability among the healthy
individuals for both ASM and GCG activities. In contrast, the affected
patients showed only residual activities of ASM (8% of mean normal
activity) and GCG (3% of mean normal activity). This is evident
from the corresponding ESI mass spectra (Figs. 1
and 2
, top panels),
which displayed much lower formation of the enzymatic products.
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The analysis of cells from affected patients also showed that
deficiencies in the activities of ASM and GCG enzymes were not
cross-correlated (Table 1
). This is evident from the ASM activities in
cell lysates from two GCG-affected patients, which were within 1 SD of
the mean of activities measured for healthy individuals. Likewise, GCG
activities in cell lysates from patients affected with ASM were within
1.2 SD of the mean of activities for healthy individuals. These results
are consistent with the previous studies of Wenger et al.
(5), who found no significant decrease of GCG activity in
cell homogenates from patients with Niemann-Pick disease.
Enzymatic rates were also measured as a function of the total cell
protein used. Both the ASM and GCG rates showed linear responses to the
amount of protein from cell homogenate as documented for ASM (Fig. 3
, top panel) and GCG (Fig. 4
, top panel). The correlation coefficients
(r2) for both the ASM and GCG
determinations were 0.998. The mean ASM activity toward the
biotinylated substrate conjugate (53 ± 12 nmol · mg
protein-1 · h-1) was
somewhat lower than that reported by Wenger (6) for
14C-labeled sphingomyelin (83 ± 23
nmol · mg-1 · h-1).
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Detection limits achieved by the ESI-MS assays can be estimated from
the amount of cellular protein used and the analyte and background peak
intensities in the ESI mass spectra. The ESI-mass spectrum shown in the
bottom panel of Fig. 1
was obtained by injecting 5–10 µL (5–10% of
the 100-µL total volume) of the product-conjugate solution used for
assaying 30 µg of protein from 
105 cells.
Pooling of this product-conjugate solution permitted 10–20 replicate
analyses to be made per assay. The product-conjugate solution volume
could be readily decreased to 5 µL by carrying out the bioaffinity
capture and release in a capillary microvessel (16),
which reduces the total cell protein needed per assay by 10- to
20-fold. The m/z 780–790 region, which contains the peaks
of the ASM product and internal standard, was free of interfering
peaks, providing a low background.
The ESI-MS data showed that at a background variation of 3
, a
16-fold reduction in the product conjugate concentration obtained
from the assay would still allow unambiguous detection of enzyme
activity >0.3
nmol · mg-1 · h-1.
Hence, the product-conjugate solution from the microvolume assay could
be diluted as needed for replicate injections and ESI-MS measurements.
We estimate that ESI-MS could allow assays using <2 µg of protein,
corresponding to <8000 cells, to be carried out routinely
(16). Because 2 x 106 cells
typically are harvested per cell culture, these can be pooled for
multiplex assays of >200 different enzymes using ESI-MS.
Detection of the GCG product and internal standard was accompanied by
somewhat higher background peaks at adjacent m/z values that
appeared consistently in replicate analyses (Fig. 2
). The origin of the
background peaks was not determined. Although the background peaks did
not overlap directly with the peaks of the analytes, they could provide
an increased background, leading to higher detection limits.
Nevertheless, the ESI-MS analysis achieved clear distinction between
the GCG product obtained from cell lysates from healthy patients (Fig. 2
, bottom panel) and that obtained from cell lysates from affected
patients (Fig. 2
, top panel). The mean activity of GCG from skin
fibroblasts toward the biotinylated substrate conjugate (5.3 ± 1
nmol · mg-1 · h-1)
was comparable to that reported for radiolabeled galactosyl ceramide
(4.7 nmol · mg
protein-1 · h-1)
(5).
Formation of both the ASM and GCG products showed a linear dependence
on the incubation time (Figs. 3
and 4
, bottom panels). These results
indicate that the total amount of enzymatic product that was generated
by either assay could be varied by adjusting the incubation time to
facilitate analysis and also that the initial enzymatic reaction
velocities were being measured.
In conclusion, we demonstrate the use of ESI-MS for assaying the lysosomal enzymes ASM and GCG in cultured skin fibroblasts. This new method does not require radioactive substrates, achieves product speciation with low backgrounds, and provides detection limits comparable to those from assays using radioactive labels. Because quadrupole ion trap and other types of compact mass spectrometers are increasingly used in biomedical research, ESI-MS-based assays can provide convenient alternatives to the currently used methods. Perhaps most importantly, because enzymatic products are resolved on the basis of their m/z ratios, it is possible to develop protocols that assay multiple enzymes in a single analysis. This can be done by multiplexing the assay procedures (16) or by multiplexing mass spectrometric analyses by combining eluates from separate enzymatic assays.
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