This document describes a new six-plex mass spectrometry method to measure enzyme activities in dried blood spots that is able to distinguish between normal and low enzyme activity samples for six lysosomal storage disorders (MPS II, MPS IIIB, MPS IVA, MPS VI, MPS VII and CLN2). The method requires a single dried blood spot, shows good precision and linearity for each enzyme, and clearly differentiates between normal and low activity samples.

1. PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com
Introduction1
5
Six-plex MS/MS method to measure I2S, NAGLU, GALNS, ARSB, GUSB and TPP1 enzyme activities in DBS
Joe Trometer, Anna Potier, Jason Cournoyer, and Mack Schermer; PerkinElmer, Waltham, MA 02451
Alyssa Vranish and Jim DiPerna; PerkinElmer, Bridgeville, PA 15017
Yang Liu, Fan Yi, Naveen Kumar Chennamaneni, Zdenek Spacil, Arun Kumar, Joyce Liao, Michael H. Gelb, C. Ronald Scott and Frantisek Turecek; University of Washington, Seattle, WA 98195
The mucopolysaccharisodes (MPS) family of lysosomal storage
disorders (LSDs) is caused by defects in the metabolic
breakdown of glycosaminoglycans (GAGs). Our previous work
demonstrated the ability to distinguish samples with low
enzyme activities for I2S (MPS II), NAGLU (MPS IIIB), GALNS
(MPS IVA) and ARSB (MPS VI) using a single 3.2 mm dried
blood spot (DBS) punch and one incubation cocktail. Adding
to this MPS family, the current work will focus on also
measuring the enzyme activity of GUSB (MPS VII) and TPP1
(CLN 2). This new six-plex still requires only a single DBS that
is incubated overnight at 37 °C followed by a post-incubation
workup that is less than 30 minutes per plate. Sample-to-
sample time using MS/MS analysis can be as low as 2 minutes,
which allows the possibility to obtain more than 4320 results
per day if desired.
Method performance studies show good linearity for each
enzyme in their respective activity range. Furthermore, a
study consisting of several hundred presumed healthy
neonates, confirmed low I2S/NAGLU/GALNS/ARSB/GUSB/TPP1
activity and CDC control DBS showed excellent resolution and
clear distinctions between the different enzyme activity levels.
Summary
Waters®, Xevo® and VanGuardTM are trademarks of Waters Technologies Corporation.
Materials and Assay Procedure2
Formulation of 96 sample S+IS vial (3.3 mL) assay cocktail
Substrates MW µM mg/vial Int. Standards MW µM µg/vial
I2S-S 767.2 500 1.19 I2S-IS 648.3 5 11
NAGLU-S 622.8 500 1.03 NAGLU-IS 422.6 5 7
GALNS-S 781.9 1000 2.52 GALNS-IS 689.9 5 11
ARSB-S 753.9 1000 2.42 ARSB-IS 661.8 5 11
GUSB-S 609.7 500 1.01 GUSB-IS 440.6 10 15
TPP1-S 638.4 200 0.42 TPP1-IS 358.3 15 18
Buffer Composition
Reagents MW mM
Ammonium Acetate (pH 5.0) 77.1 60
Cerium Acetate 317.3 7
NAG-thiazoline 219.3 0.1
MSMS
3.2 mm DBS punch
to 96-well plate
Quench & Mix
37°C, 400 rpm
18 ± 2 hours
Remove seal, add 100
µL
50:50 MeOH:EtOAc
Mix with pipette
Transfer Liquid & Extract
Transfer Top
Layer
Transfer 200 µL top layer
to sampling plate
Room temp
(25°C) at
400 rpm 10
min
Shake &
Incubate
Transfer to 96 deep well plate
(DWP)
Add 400 µL EtOAc then 200 µL 0.5
M NaCl Solution Mix with pipette
Separate Layers
Centrifuge DWP 5 min,
700 x g
Dry
Evaporate at
40°C, 10-15 min
Reconstitute with 100
µL 60:40 H2O:ACN with
0.1% formic acid
Add Flow Solvent
Add 30 µL
cocktail Seal plate
Punch
Shake
Add Cocktail
1 2 3 4
56789
10uL injection
using X-Select
column
The assay procedure is detailed in the workflow diagram in
Figure 1. The formulation of the incubation cocktail is in Table
1 and the structures of the substrates and IS are in Figure 2.
Table 1. 6-plex incubation cocktail components
Figure 1. 6-plex Assay Protocol
Substrates Internal Standards
Samples were analyzed using a Waters® Xevo®
TQD with an X-select CSH C18 column and a
VanGuardTM CSH precolumn. A flow solvent
consisting of 60:40 H2O:Acetonitrile with 0.1%
formic acid at 0.5 mL/min showed good
separation between the internal standard and
residual substrate peaks for each analyte.
To assess assay performance, a study consisting
of DBS from de-identified presumed healthy
subjects (N=616) collected in August 2016 and
DBS with confirmed low (N=29) activity for I2S,
NAGLU, GALNS, ARSB, GUSB and TPP1 were
run over 3 days (~3 plates per day). Each plate
had 2 blanks (punched blank filter paper) and
two replicates of each control level. Assay
precision was estimated using the 18 control
replicates each of low/mid/high over the 3
days.
Methods3
Dot plots of the results for the six enzymes are
shown in Figure 3. Multiple replicates (N=18) of
the low, mid and high control DBS showed good
precision in the assay for all six enzymes. Table
2 shows that all controls had %CV ≤ 11%
except the low control for NAGLU and TPP1.
The data shows clear distinction between the
presumptive normal and the confirmed low
activity DBS suggesting that a cut off can easily
be made to routinely differentiate low activity
from normal neonatal DBS. Furthermore, all
confirmed low activity samples were less than
10% of the average presumptive neonatal
population and also less than the lowest
presumptive normal activity for each enzyme,
as shown in Table 2.
Results4
A new tandem mass spectrometric multiplex assay for
measuring I2S, NAGLU, GALNS, ARSB, GUSB and TPP1 enzyme
activities in DBS was demonstrated. This method was able to
clearly differentiate between the activities in samples collected
from presumed healthy subjects and in DBS samples from
individuals confirmed to have low enzyme activity. Good
precision and linearity of each enzyme was demonstrated using
control DBS.
• We thank the Mayo Clinic, Greenwood Genetics and Serv.
Genet. Med. HCPA for providing confirmed low activity DBS
samples.
All DBS samples were collected following IRB approved
protocols.
Type
Enzyme Activity (mM/hr)
I2S NAGLU GALNS ARSB GUSB TPP1
Low Control 0.78 0.21 0.46 1.68 8.51 5.33
%CV 7% 24% 9% 10% 11% 22%
Mid Control 6.89 2.68 3.04 11.6 25.7 19.9
%CV 5% 10% 6% 8% 7% 10%
High Control 13.0 5.85 6.14 24.3 45.1 24.9
%CV 5% 10% 6% 11% 7% 6%
Ave. Normal Neonatal 13.8 2.56 2.52 11.0 27.7 20.4
Lowest Normal Neonatal 6.74 1.17 0.72 3.33 14.8 11.6
Highest Confirmed Low 1.22 0 0.24 1.13 0.11 0.64
Table 2. Average activities for control DBS and DBS from presumptive normal neonatal
DBS for the enzymes I2S, NAGLU, GALNS, ARSB, GUSB and TPP1. The lowest normal
neonatal is the lowest activity measured of the 616 DBS analyzed and the highest
confirmed low is the highest measured activity in the confirmed low-activity cohort.
Figure 3. Dot plots for the enzymes measured in DBS from the presumptive normal
neonatal, DBS from individuals with confirmed low enzyme activity and low, mid and
high controls.
R² = 0.995
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
TheoreticalEnzymeActivity(mM/hr)
Observed Enzyme Activity (mM/hr)
I2S
R² = 0.9967
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50
TheoreticalEnzymeActivity(mM/hr)
Observed Enzyme Activity (mM/hr)
NAGLU
R² = 0.9955
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
TheoreticalEnzymeActivity(mM/hr)
Observed Enzyme Activity (mM/hr)
GALNS
R² = 0.9911
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00
TheoreticalEnzymeActivity(mM/hr)
Observed Enzyme Activity (mM/hr)
ARSB
R² = 0.9961
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
8.00 13.00 18.00 23.00 28.00 33.00 38.00 43.00 48.00
TheoreticalEnzymeActivity(mM/hr)
Observed Enzyme Activity (mM/hr)
GUSB
R² = 0.9804
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00
TheoreticalEnzymeActivity(mM/hr)
Observed Enzyme Activity (mM/hr)
TPP1
Figure 4. Linearity series for the enzymes measured in DBS.
I2S
NAGLU
GALNS
ARSB
GUSB
TPP1