
We report the development of MS/MS assays for I2S, GALNS, and ARSB that give a much higher assay response in the mass spectrometer than previously reported assays. These new reagents lead to a larger lysosomal enzyme assay analytical range, which we defined as the ratio of assay response with the high QC DBS, because of the relevant enzymatic reaction, divided by the response for nonenzymatic processes. Increasing the analytical range is important for NBS and diagnosis of LSDs because this is predicted to lead to a more accurate enzyme activity value at the low end. This is expected to lead to better differentiation between disease-affected patients and those with pseudodeficiencies and, in general, lead to a lower rate of false positives. For diagnosis, it may lead to better prediction of disease severity. We also compared the analytical range of 6 MS/MS assays to those measured fluorometrically with 4MU-substrates.
Methods
All methods, including the synthesis of the substrates, are described in the Data Supplement that accompanies the online version of this report at http://www.clinchem.org/ content/vol61/issue11.
Results

FIA-MS/MS ASSAYS FOR GALNS AND ARSB
Our original MS/MS substrate for GALNS consisted of a Gal-6-sulfate linked to an analog of 4MU bearing a hydrophobic chain (8 ) . Although this assay distinguished between healthy and MPS-IVA samples, the MS/MS signal for the product in assays with random newborns was 50-fold less than for our other MS/MS assays for lysosomal enzymes. This original assay is not sufficiently robust for NBS, and the only way forward is to find a higher activity substrate or to increase the MS/MS response of the product. GALNS is thought to be responsible for removal of sulfate from Gal-6-sulfate and GalNAc-6-sulfate in mucopolysaccharides (4 ) . Thus, we explored the consequence of replacing Gal-6-sulfate in our previous substrate with GalNAc-6-sulfate. We also replaced the 4MU-based aglycone in our original GALNS substrate with an aglycone containing the 4-acetamidophenol moiety because this aglycone change results in an improved assay MS/MS response per mole for the product derived from our new I2S substrate (6 ) . The structure of our new GALNS substrate, GalNAc-6-S-C6/C6-benzoyl group (Bz), is shown in Fig. 1 . The name derives from the presence of the N-hexanoyl group (C6), the hexamethylene linker (C6), and the Bz in the aglycone. Calculations suggest that the 2 carbonyl groups in the aglycone serve as a site of facile protonation (Fig. 1) , which allows for higher-yield ionization in the electrospray source (11 ) . We also pursued new assays for ARSB using GalNAc-4-S-C5/C5-Bz (Fig. 1) .
FIA-MS/MS ASSAYS FOR GALNS AND ARSB
Online Supplemental Table 3 shows that the k cat /K m for GalNAc-6-S-C6/C6-Bz was 81-fold larger than that for Gal-6-S-C7/C6-Bz, indicating that GALNS greatly preferred the GalNAc substrate (we did not have the comparator compound Gal-6-S-C6/C6-Bz, but it is unlikely that GalNAc distinguishes between the number of methylenes in the aglycone). These studies justified the selection of our new GALNS GalNAc-6-S-C6/C6-Bz for continued studies.
FIA-MS/MS ASSAYS FOR GALNS AND ARSB
The first version of the MS/MS assay was based on flow injection analysis (FIA). We considered the possibility of adding an enzyme that cleaved the glycoside only after the sulfatase removed the sulfate, thus generating the aglycone (Fig. 2) . If the aglycone product gave a stronger MS/MS response per mole than that for the immediate sulfatase product (desulfated glycoside), use of the coupled enzymatic process would yield a gain in analytical range. Furthermore, buffer salt removal by liquid-liquid extraction was required for FIA-MS/MS to prevent suppression during the ionization process. In the case of sulfatases the initial product contains the sugar, which is hydrophilic and reduced the solvent extraction yield. Conversion to the aglycone will increase the transfer of analyte to the organic layer.
FIA-MS/MS ASSAYS FOR GALNS AND ARSB
The ion response per mole of the GALNS-and ARSB-generated aglycones, C6/C6-Bz and C5/C5-Bz, were both found to be 2.4-fold larger than that for the corresponding glycosides GalNAc-6-S-C6/C6-Bz and The proton shown hydrogen bonded to the 2 carbonyl groups is the proposed site of gas phase protonation in the electrospray ion source. Internal standards lack the sulfate and contain 5 deuteriums on the benzoyl group.
FIA-MS/MS ASSAYS FOR GALNS AND ARSB
GalNAc-4-S-C5/C5-Bz. This finding, along with the ethyl acetate consideration, argued for use of a coupled assay with enzymatic removal of the desulfated sugar. A disadvantage of this approach became apparent when we studied the pH dependence of the nonenzymatic hydrolysis of GalNAc-6-sulfate-C6/C6-Bz to the aglycone (see online Supplemental Fig. 1 ). Glycoside hydrolysis in the absence of DBS increased with a drop in pH, showing that it was acid catalyzed. It is known that glycosides of 2-acetamido sugars are more acid sensitive than those with a 2-hydroxyl because of neighboring group participation by the acetamido group (12 ) . This acid-catalyzed pathway gave rise to aglycone, thus increasing the background of the assay. With DBS, enzymatic formation of the sulfatase product GalNAc-C6/C6-Bz was maximal at pH approximately 5.0, and thus going to pH Ͼ5.0 would not help. Similar pH-dependent observations were seen with the ARSB substrate GalNAc-4-S-C5/ C5-Bz (see online Supplemental Fig. 1 ).
FIA-MS/MS ASSAYS FOR GALNS AND ARSB
The use of GalNAc-4-S-C5/C5-Bz and GalNAc-6-S-C6/C6 required complete inhibition of hexosaminidase A because this enzyme can cleave the glycoside of GalNAc residues containing a 6-sulfate (13 ) ( Fig. 2 ; also see online Supplemental Table 4 ). Recombinant human hexosaminidase A cleaved GalNAc-4-S-C5/C5-Bz, showing that the enzyme tolerated 4-sulfation (see online Supplemental Table 5 ). Endogenous hexosaminidase A present in DBS cleaved these glycosides (see online Supplemental Table 5 ). The hexosaminidase A inhibitor (Z)-Pugnac (14 ) at 1 mmol/L blocked almost all of the activity of recombinant hexosaminidase A and enzyme in DBS (see online Supplemental Table 6 ). In subsequent assays we used 2 mmol/L (Z)-Pugnac to ensure complete hexosaminidase A inhibition. To generate the aglycone from the desulfated glycoside, we explored the bacterial enzyme ␤-N-acetylgalactosaminidase (␤-NGA) (15 ) . The data in online Supplemental Tables 6 and 7 show that ␤-NGA did not generate aglycones from the sulfated substrates, and ␤-NGA catalyzed hydrolysis of the nonsulfated GALNS and ARSB products were not sensitive to (Z)-Pugnac. This allowed us to develop the new FIA-MS/MS assay of GALNS and ARSB based on the natural GalNAc-4/6-sulfate substrates (described above).
FIA-MS/MS ASSAYS FOR GALNS AND ARSB
The internal standard for GALNS is GalNAc-C6/ C6-Bz with 5 deuteriums in the benzoyl group. Thus, any incomplete ␤-NGA reaction is accounted for by us-
Fig. 2. Enzymatic reactions for I2S, GALNS, and ARSB.
Coupling enzymes to convert the direct product of the lysosomal enzyme to the free aglycone are shown (this step is omitted for some variations of the assays described in the main text). The use of (Z)-Pugnac to block the action of human hexosaminidase A on the GALNS and ARSB substrates is also shown.
Fig. 2. Enzymatic reactions for I2S, GALNS, and ARSB.
ing the glycoside internal standard. We found that increasing the amount of ␤-NGA beyond that in our standard assays did not increase the overall rate of aglycone formation (data not shown), showing that the reaction was limited by the sulfatase as desired. Selective reaction monitoring was used with FIA-MS/MS to measure the amount of nondeuterated product aglycone and deuterated internal standard aglycone after collision-induced dissociation of precursor ions. Table 1 lists the analytical range of the FIA-MS/MS assays for GALNS and ARSB. The analytical range was defined as the ratio of assay response for the QC high sample due to the relevant enzyme divided by the response for nonenzymatic processes (calculated as described in the online Data Supplement).
LC-MS/MS ASSAYS FOR GALNS AND ARSB
Given the multiple factors that contribute to analytical range in the FIA-MS/MS assay using ␤-NGA, we also studied a second assay for GALNS and ARSB based on coupled LC-MS/MS. In this case, sample desalting occurred on the liquid chromatography (LC) column, and thus conversion of the glycoside product to the aglycone with ␤-NGA and liquid-liquid extraction with ethyl acetate were not required. The assay incubation was quenched with acetonitrile, and precipitated macromolecules were pelleted by centrifugation. The supernatant was diluted with water and subjected to LC-MS/MS. Because we detected the desulfated products GalNAc-C5/C5-Bz and GalNAc-C6/C6-Bz rather than the aglycones, we were not concerned with acid-catalyzed glycoside hydrolysis. We were also not concerned with enzyme-independent desulfation of substrates in the electrospray ion source because substrate and product had distinct LC retention times (see online Supplemental Fig.  2 ), and we integrated only the region of the chromatograph in which product and internal standard eluted. (Z)-Pugnac was still added because a substantial amount of the internal standard was cleaved without the hexosaminidase A inhibitor (not shown). To minimize the instrumental complexity, we used a normal-pressure LC column and developed an isocratic solvent system to separate the GALNS and ARSB products from their corresponding substrates. In this way we simply inserted the LC column into the solvent delivery system that was used for FIA-MS/MS. Furthermore, in our previous studies of LC-MS/MS for LSD assays (16 ) , several thousand assays could be obtained without column replacement. Table 1 lists the analytical ranges for the GALNS and ARSB LC-MS/MS assays. Given the higher analytical ranges, we carried out additional studies using LC-MS/MS rather than FIA-MS/MS assays. To demonstrate the reproducibility of the assay and the linearity with respect to the amount of enzyme in the DBS, we carried out 5 independent replicate assays on a series of QC DBS containing various amounts of leukocyte-depleted and whole blood (Table 1 ; linearity plots provided in online Supplemental Fig. 3) .
LC-MS/MS ASSAYS FOR GALNS AND ARSB
Assay results for 50 random newborns and MPS-IVA-and MPS-VI-affected patients are provided in Fig.  3 (tabular data in online Supplemental Tables 8 and 9 ), showing that this LC-MS/MS assay readily distinguished between 50 healthy newborns and MPS-IVA or MPS-VI patients.
MS/MS ASSAYS FOR I2S
We explored a variation of our previous MS/MS assay for I2S by adding IDUA to generate the aglycone after desulfation (Fig. 2) 
TRIPLEX ASSAY FOR MPS-II, -IVA, AND -VI
After exploring several possible options, we put all 3 MPS assays into a single triplex assay using a single cocktail incubated with a single 3-plex DBS punch. For solubility reasons we reduced the concentration of the MPS-II substrate to 0.5 mmol/L and kept the other 2 substrates at 1 mmol/L (I2S had the highest activity). We included citric acid in the quench step so that the MPS-II product containing the iduronic acid group would protonate on its carboxylate to facilitate extraction into ethyl acetate during the liquid-liquid extraction. The extract was submitted to LC-MS/MS using the above isobaric conditions under low pressure. Ethyl acetate extraction was found to provide a more robust assay because it led to a large reduction in the amount of substrate in the mixture applied to the LC column, which in turn reproducibly ensured complete separation between substrates and product/internal standards. Results are summarized in Table 2 , and linearity data using QC standards is shown in online Supplemental Fig. 3 .
ANALYTICAL RANGE FOR FLUOROMETRIC ENZYME ASSAYS BASED ON 4MU SUBSTRATES
We used the 4MU substrates in Table 3 to assay lysosomal enzymes IDUA, acid ␣-glucosidase, I2S, GALNS, and ARSB. The principle of these assays is based on enzymatic release of 4MU from the substrate conjugate (i.e., 4MU-glycosides). The mixture was quenched with a buffer with a pH of approximately 10 to cause ionization of the hydroxyl group of 4MU to its phenolate (4MU-OH to 4MU-O Ϫ ). The pK a of 4MU-OH is 7.6, 16 (17 ) . We confirmed these findings by spectral titration of 4MU (not shown). The above analysis showed that the neutral species 4MU-OH had finite fluorescence, and thus we explored the fluorescence of 4MU-glycoside substrates. Table 3 lists the percentages of 4MU present as an impurity in the substrates and the ratio (4MU-O Ϫ emission per mole)/(4MU-glycoside emission per mole). These ratios were in the range 2200 -5400 (the outlier was for 4MU-sulfate, ratio ϭ 58 700) and accounted for the increase in fluorescence when 4MU-glycosides were acted on by lysosomal enzymes. Although this ratio was much higher than 1, in assays with DBS, the percentage of total 4MU-substrate that was converted to 4MU was typically only approximately 1%. Thus, the intrinsic fluorescence of the 4MU-glycoside substrate greatly increased the background and reduced the analytical range.
ANALYTICAL RANGE FOR FLUOROMETRIC ENZYME ASSAYS BASED ON 4MU SUBSTRATES
The analytical ranges of these 4MU assays were defined as above (details of the calculation provided in the online Data Supplement) and listed in Table 3 . Background-corrected enzyme activities are also listed in Table 3 .
Discussion ANALYTICAL RANGE FOR 4MU-FLUOROMETRIC VS MS/MS ASSAYS OF LYSOSOMAL ENZYMES
For the 6 4MU assays studied, the analytical ranges were in the range 6 -16 (except 34 for ARSB with 4MU-sulfate) (Table 3) when measured with the same QC high DBS (PerkinElmer QC High, see Methods in the online Data Supplement). For all of the assays shown in Table 2 , the fluorescence from the buffer or the buffer incubated with the DBS punch, both lacking substrate, was Ͻ5% of the fluorescence measured with buffer containing 4MU-glycoside substrate (data not shown). What limits the analytical range of these assays is clearly the high background due to the intrinsic fluorescence of the 4MU-glycoside substrates and the small fraction of substrate converted to product.
Discussion ANALYTICAL RANGE FOR 4MU-FLUOROMETRIC VS MS/MS ASSAYS OF LYSOSOMAL ENZYMES
The analytical ranges for the MS/MS assays were much larger than those for 4MU assays, 51-fold for I2S and 30-fold for GALNS. For ARSB, the MS/MS analytical range was 6.6-fold higher than that for the 4MU assay if 4MU-sulfate was used or 29-fold if the more natural-like substrate 4MU-GalNAc-4-S was used (see below for concerns about using 4MU-sulfate). We hypothesize that the assays with higher analytical range will lead to a better separation between nonaffected and LSDaffected individuals, which will lead to lower falsepositive rates in NBS and to more accurate estimation of disease prognoses. Comparison of large-scale NBS pilot studies has already shown evidence that the MS/MS method leads to substantially lower false positives (18 -20 ) .
GALNS ASSAYS
The originally reported assay for GALNS used 4MU-Gal-6-sulfate (21 ) and leukocytes as the enzyme source. This assay relied on endogenous human ␤-galactosidase to release the 4MU after desulfation by GALNS. Extension of this assay to DBS involved supplementation of the cocktail with Apergillus oryzae ␤-galactosidase (Sigma Chemicals) that is active at pH approximately 4 (7 ). We attempted this GALNS assay with DBS and found that the commercial Apergillus oryzae ␤-galactosidase from Sigma was capable of generating 4MU from 4MU-Gal-6-sulfate (no difference in assay response was observed for assays with DBS vs filter paper, data not shown), suggesting that this preparation of enzyme is contaminated with a 6-sufatase. The calculation of the analytical range of this GALNS assay with DBS was not possible because the assay responses for the blanks were not stated (7 ) . However, we can say that the mean GALNS activity measured for 50 random newborn DBS with 3 mmol/L 4MU-GalNAc-6-S is 3.
GALNS ASSAYS
reported using 10 mmol/L 4MU-Gal-6-S (7 ). This suggests that GALNS is approximately 44-fold more active on 4MU-GalNAc-6-S vs 4MU-Gal-6-S, which is consistent with our MS/MS data (see online Supplemental Table 3 ). Thus the analytical range using 4MU-Gal-6-S is likely less than the value of 6.5 for 4MU-GalNAc-6-S. These relatively low analytical ranges suggest that the 4MU assays for GALNS will not be appropriate for NBS of MPS-IVA. The enormous increase in analytical range for the LC-MS/MS assay vs the a Values calculated as described in the online Data Supplement. QC samples from the CDC are base pool (B, 0% whole blood), low (L, 5% whole blood), medium (M, 50% whole blood), and high (H, 100% whole blood). Same for the PerkinElmer (PE) samples except there is no base pool. fluorometric assay for GALNS is due mainly to the factors described above that limit the analytical range of the 4MU assays rather than a difference in the enzyme activ-
GALNS ASSAYS
) for the MS/MS vs 4MU substrates (the same is true for the ARSB and I2S assays, see below).
GALNS ASSAYS
The GALNS FIA-MS/MS method requires the use of ␤-NGA and ethyl acetate extraction. The analytical range is 4-fold better than that for the 4MU-GalNac-6-S fluorometric assay (Tables 1 and 2) , and the LC-MS/MS assay in the absence of ␤-NGA and ethyl acetate is 8.5-fold better than the FIA-MS/MS assay and provides a high-performance GALNS assay ( Table 2 ). Product and ion counts are approximately 200-fold higher than those of our original GALNS MS/MS assay based on Gal-6-S (8 ). This assay readily distinguished between 50 healthy newborns and the MPS-IVA patients (Fig. 3) . Pilot studies using this new GALNS assay (and ARSB assay, see below) have started in the Washington State Department of Health NBS laboratory, which seems timely given that enzyme replacement treatment is now available (23 ) . Adding a low-pressure LC column with an isocratic elution solvent does not add significant instrumentation complexity because the low pressure column is compatible with currently used FIA solvent delivery systems and column cost is insignificant.
ARSB ASSAYS
The established assay for ARSB for diagnosis of MPS-VI uses the nonspecific sulfatase substrate 4MU-sulfate or related catechol sulfates (24 ) . Studies with DBS from MPS-VI patients show low but residual ARSB activity, suggesting that this substrate is detecting mainly ARSB. The source of this residual activity is unknown. It may be due to residual activity of ARSB or due to other sulfatases. Of interest is arylsulfatase A, the enzyme deficient in metachromatic leukodystrophy, because it hydrolyzes 4MU-sulfate (4, 25 ) . Arylsulfatase A is active in samples using DBS because it can be detected after immunoprecipitation and subsequent assay with 4MU-sulfate (25 ) . Additional studies show that this enzyme is relatively unstable in DBS (25 ) . All together the studies strongly suggest that the use of the nonspecific sulfatase substrate 4MU-sulfate is not appropriate for NBS of MPS-VI in which fresh DBS will be the enzyme source. Caution is also warranted for diagnostic assays using DBS or leukocyte samples that have not been confirmed to lack arysulfatase A activity. The distinction between ARSB and arylsulfatase A using 4MU-sulfate can be improved by using different buffer conditions for the 2 assays (4 ), but there is still considerable crosstalk between these 2 assays (4 ).
ARSB ASSAYS
The ARSB FIA-MS/MS assay described here uses ␤-NGA, hexosaminidase A inhibitor, and ethyl acetate extraction. The analytic range is comparable to the fluorometric assays with 4MU-sulfate and 3-fold better than the fluorometric assay with 4MU-GalNAc-4-S. Again, the LC-MS/MS assay without coupling enzyme is superior, being 10-fold better in analytical range than the FIA-MS/MS assay. The assay distinguishes healthy newborns from MPS-VI patients (Fig. 3) .
I2S ASSAYS
The analytic range of the FIA-MS/MS assay is 34-fold higher than that with the 4MU-IdA-2 assay and is improved another approximately 2-fold if we use LC-MS/MS without the need for adding IDUA-coupling enzyme. A pilot study using the MS/MS I2S assay for NBS of MPS-II has begun in the Washington State Department of Health NBS laboratory.
TRIPLEX ASSAYS
Combining all 3 sulfatase assays into a single triplex assay using a single assay cocktail and a single 3-mm DBS punch was straightforward, and results shown in Table 2 and online Supplemental Fig. 3 demonstrate that the method works essentially as well as for the individual assays, again with analytic ranges that greatly exceed those seen with 4MU assays.

{'paper_id': '25457146', 'title': 'Tandem Mass Spectrometry Has a Larger Analytical Range than Fluorescence Assays of Lysosomal Enzymes: Application to Newborn Screening and Diagnosis of Mucopolysaccharidoses Types II, IVA, and VI', 's2_url': 'https://api.semanticscholar.org/CorpusID:25457146', 'source': 'S2ORC'}