Dataset and standard operating procedure for newborn screening of six lysosomal storage diseases: By tandem mass spectrometry

In this data article we provide a detailed standard operating procedure for performing a tandem mass spectrometry, multiplex assay of 6 lysosomal enzymes for newborn screening of the lysosomal storage diseases Mucopolysaccharidosis-I, Pompe, Fabry, Niemann-Pick-A/B, Gaucher, and Krabbe, (Elliott, et al., 2016) [1]. We also provide the mass spectrometry peak areas for the product and internal standard ions typically observed with a dried blood spot punch from a random newborn, and we provide the daily variation of the daily mean activities for all 6 enzymes.


Specifications
Value of the data Standard operation procedure gives full "hands-on" instructions for laboratory workers with appropriate training to carry out the 6-plex tandem mass spectrometry assay for lysosomal storage diseases.
Raw data for the assays are provided so that other laboratories can compare their raw data to that given in this publication.
Data is useful for setting up the new mass spectrometry assays in newborn screening laboratories including troubleshooting.

Data
Data provided are: 1) Fig. 1 provides the enzymatic activity (μmole/h/L blood) for each of 6 lysosomal enzyme activities averaged across all random newborn samples (data obtained according to the standard operating procedure given below). The mean activity is provided as a function of assay date. 2) Table 5 gives in peak areas for the multiple-reaction monitoring ion chromatograms for each of the 6 enzymatic products and internal standards observed with the quality control HIGH standard (typical of a healthy newborn). 1. Dissolve 5.1 mg acarbose, 11.18 g N-acetylgalactosamine, 8.73 mg D-saccharic acid 1,4-lactone monohydrate, 14.80 g sodium taurocholate, 10.50 g succinic acid, and 82 mg of zinc chloride in nearly 1 L of purified water (Milli-Q, Millipore Corp. or other LC-MS/MS grade water). 2. Use a pH meter freshly calibrated with pH 4.0 and pH 7.0 buffers, and bring the pH of the mixture to 4.71 using sodium hydroxide. Finally, add water to bring to 1 L. 3. Store the buffer in a plastic bottle (PET or PP material) at þ 2 to þ8°C for up to 12 months.

Substrate/internal standard mix
This is available form PerkinElmer (1 vial for ten 96-well plates). It contains a mixture of 6 substrates, 6 internal standards, and sodium oleate. The dried mix should be kept at À15 to À30°C and can be stored for up to 1 year. Sodium oleate is a component to promote higher activity of the enzymes that act on sphingolipids. It is important to remember that it is supplied as a component of the substrate/internal standard mix, and thus is not added at the time of buffer preparation.
1.4. 6-plex assay cocktail 1. Add 33 ml of the 6-plex buffer to one vial of 6-plex substrate/internal standard vial. 2. Sonicate for 20 min in a bath-type sonicator. Swirl and invert the vial several times. Do not vortex as this will produce a foam. A stir bar and stir plate can be used instead. Repeat sonication if necessary until all substrate is dissolved into a clear solution. Table 4 below gives the composition of assay cocktail. 3. Wrap in foil (light sensitive), store vial at room temperature for up to 1 week (or up to 1 month at þ 4°C).

6-plex quench solution
Prepare in chemical hood. Add 500 mL of ethyl acetate to 500 mL of methanol, swirl to mix, store in hood at room temperature for up to 6 months in a glass bottle.
The quality of ethyl acetate used in this step as well as the liquid-liquid extract step (day 2 sample work-up) should be considered. Trace amounts of oxidizers (e.g., peracetic acid) from ethyl acetate manufacturing have been found to lower the product and internal standard intensities for GLA, GAA and IDUA. The lowering of these signals can affect the accurate measurement of low-activity samples. HPLC and LC-MS/MS grade ethyl acetate from J.T. Baker (Avantor) have been found to be routinely of good quality for this application (undetectable amount of oxidizers). Ethyl acetate with trace oxidizer contamination can cleaned by treatment with anion exchange resin (such as Dowex-1). If needed you can swirl 10 g of Dowex-1 in an Erlenmeyer flask with 50-100 mL of ethyl acetate, then decant, repeat 3 times (this is to remove contaminants which may be present on the surface of commercial Dowex-1). Then transfer the washed Dowex-1 to a glass bottle of ethyl acetate (1-2 L), swirl briefly, and then use for the assay. There is no need to remove the Dowex-1 beads, they will remain at the bottom of the bottle.
Methanol used should be LC-MS/MS grade.
1.6. 6-plex mobile phase 84% acetonitrile/16% water/0.1% formic acid 1. Measure out 320 mL of HPLC grade water (Fisher Optima Grade) in a graduated cylinder then transfer to a 2 L volumetric flask. 2. In a chemical hood, add 2 mL of HPLC grade formic acid (Fisher Optima Grade) to the volumetric flask, then swirl to mix. 3. In a chemical hood, QS the volumetric flask to 2 L with HPLC grade acetonitrile (Fisher Optima Grade). 4. Invert to mix. 5. Store at room temperature for up to 3 months. will proceed with QC DBS from just a single supplier so that more newborn samples can be run per plate. 3. Fill a small narrow trough with approximately 10 mL of 6-plex assay cocktail. This will be enough for 3 plates.
4. To each well, add 30 μL assay cocktail using a multi-channel pipette. Place the tip of the pipette against the inside wall of the well when dispensing to allow cocktail to slide down the wall of the well for accurate delivery. 5. Seal plate with aluminum sealing film (StarSeal sealing tape aluminum foil, Star Lab Cat. E2796-9792, Hamburg, Germany), press firmly to ensure each well is sealed or liquid will be lost due to evaporation during incubation. You can use a sealing roller. These sealing films are not sold in the USA, but we have tested Axygen foil covers (Cat. PCR-AS-200 or VWR Cat. 47734-817), which work fine. 6. Place plates in the PerkinElmer Trinest incubator and incubate for 18 h (7 15 min) at 37°C 70. 5 with orbital shaking at 400 rpm. Make note of start time and temperature on the assay logsheet. 6. Collect 100 μL of quench solvent from the trough, move liquidator head to sample plate and dispense the 100 μL from the tips into the samples wells.
7. Mix the sample with quench solvent as follows: press tips against side wall of sample well when entering the sample well. You will have to pull the plate towards you using the liquidator plate platform. When the tips reach the bottom of the well push plate away from you. This action uses the tips to move the DBS punch to the side wall of the sample well and out of the way of the pipette tip.
Aspirate 100 μL of the sample volume up and down into the liquidator tips 10 Â .  times in each well. This is a critical step to ensure entire sample volume was miscible for a short period of time, and thus the sample is extracted from the water layer into the ethyl acetate layer.
The two layers will separate out quickly. Use a new box of tips for each plate. 8. Seal the plates with self-adhesive foil (same type of adhesive foil used for incubation). 9. Centrifuge the plates at 2500 rpm for 5 min at room temperature to separate solvent layers. 16. Transfer and dispense liquid into the corresponding shallow well plate on the right. Move quickly to the right plate as ethyl acetate has low viscosity and can drip from the tips. 17. Evaporate the solvent from the shallow plates with jets of oil-free air using a SPE 96 Dual Dryerflow rate 60-40 L/min, temperature 35°C, drying time 10 75 min.
Note, if you don not use the Liquidator you can obtain from PerkinElmer (jason.cournoyer@perkinelmer.com) a plastic multi-well spacer with holes that is placed on top of the deep well plate. The pipet tips on the multi-channel pipet are inserted into the holes in the spacer and lowered until the tips stop in the spacer. In this way the tips are lowered into the ethyl acetate layer but not into the lower water layer. It is important to use the proper tips as recommended by PerkinElmer. In this way, the tips are placed deeply enough into the ethyl acetate layer such that no air or water are drawn in the tips while 100 mL of ethyl acetate is drawn into the tips. If you don't have this spacer, you can do a trial liquid transfer where you use the indentation marks on the pipet tips to gauge how far the tips are inserted into the deep well plate. Also, an automated liquid handler can be set to be used for this step.
3. Shake each plate for 10 min in the PerkinElmer Trinest incubator without heat and at a speed of 400 rpm to dissolve the residue. Keep plates level to prevent cross-well contamination. 4. See the next section on preparing the mass spectrometer for sample analysis before injecting samples.

5.
Inject 15 μl per sample using a 10 μl loop. The effective volume of sample delivered is 10 μL as the loop is overfilled. Needle wash and flow-injection conditions for the autosampler are given below. 6. Samples are injected on the MS toward the end of day two and throughout the night.

Mass spectrometry
We have tested the 6-plex assay successfully on 5 types of instruments: Acquity TQD, Xevo TQD and Quattro Micro from Waters and the 3200 and 4000 from Sciex. The assay requires low-energy source conditions in order to minimize in-source fragmentation of the excess substrate that remains in the sample that is injected into the MS system. Under high-energy conditions, the substrate can breakdown to generate the enzymatic product and therefore can increase the measured activity in blanks and samples. Despite blank subtraction from DBS samples, it has been found that this insource decay of the excess substrate can affect the accurate measurement of low activity samples and therefore these low-energy sources conditions are required for optimum assay performance. Even with this contribution to the blank, the ratio of assay response for the quality control high DBS (typical of a healthy newborn) to response from the no-blood blank is an order-of-magnitude higher than the analogous ratio for fluorimetric assays with 4-methylumbelliferyl substrates as discussed in the main text.
The necessary low-energy conditions can primarily be achieved by lowering the temperature in the source, but also by lowering entrance voltages (i.e., cone voltages for Waters instruments and declustering potentials for Sciex instruments) and the capillary voltage if necessary. See Table 2 below for examples of low-energy source settings for the Waters and Sciex instruments that were used successfully for the assay. The final settings should be determined using blanks and obtaining the lowest signal for product (from in-source fragmentation in blanks) but also keeping the IS signal as high as possible.
As mentioned, in addition to low source temperature, the optimized entrance voltages for the MRMs can be lowered to decrease the apparent activity of the blanks. This approach has shown to be useful for keeping GBA and GALC blanks low. Also, in some cases, lowering the capillary voltage has been found to be useful for keeping GLA blanks low.
The entire analytical method uses the MS/MS settings in Table 1, MRM transitions in Table 2, and inlet and autosampler settings in Table 3.