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Multiplexing Iduronate-2-Sulphatase (MPS-II) into a 7-Plex Lysosomal Storage Disorder MS/MS Assay Using Cold-Induced
Elya Courtney1, C Austin Pickens1, Carla Cuthbert1
1Centers for Disease Control and Prevention, Atlanta, GA 30341, USA.
Abstract:
Mucopolysaccharidosis type II (MPS-II, Hunter syndrome, OMIM:30990) is a lysosomal storage disorder (LSD) that results in iduronate 2-sulphatase (I2S) enzyme deficiency. MPS-II was added to the Recommended Uniform Screening Panel (RUSP) in August 2022; thus, there is an increased demand for multiplexing I2S into existing LSD screening assays. After incubation with LSD synthetic substrates, extracts are cleaned using liquid-liquid extraction with ethyl acetate or protein precipitation using acetonitrile (ACN). We investigated cold-induced water ACN phase separation (CIPS) to improve the combination of 6-plex and I2S extracts to create a 7-plex assay, and compared it to room temperature ACN and ethyl acetate liquid-liquid extraction. The extracts were dried and resuspended in the mobile phase, and then analyzed using an optimized 1.9 min injection-to-injection liquid chromatography method coupled with tandem mass spectrometry (LC-MS/MS). The combination of ACN and CIPS improved the detection for I2S products without significant detriment to other analytes, which is attributable to a more complete coagulation and separation of heme, proteins, and extracted residual salts. Using CIPS for sample cleanup in dried blood spots (DBS) appears to represent a promising and straightforward way of achieving cleaner sample extracts in a new 7-plex LSD screening panel.
Insights
Mucopolysaccharidosis type II (MPS-II) screening is crucial. Cold-induced water acetonitrile phase separation (CIPS) enhances sample cleanup for multiplexed lysosomal storage disorder (LSD) assays, improving iduronate 2-sulphatase (I2S) detection.
Area of Science:
- Biochemistry
- Mass Spectrometry
- Newborn Screening
Background:
- Mucopolysaccharidosis type II (MPS-II), or Hunter syndrome, is a lysosomal storage disorder (LSD) caused by iduronate 2-sulphatase (I2S) deficiency.
- MPS-II inclusion on the Recommended Uniform Screening Panel (RUSP) necessitates improved multiplexed screening assays for LSDs.
Purpose of the Study:
- To evaluate cold-induced water acetonitrile phase separation (CIPS) as a sample cleanup method for a 7-plex LSD screening assay, specifically enhancing I2S detection.
- To compare CIPS with traditional ethyl acetate liquid-liquid extraction and room temperature acetonitrile precipitation for LSD screening.
Main Methods:
- Investigated CIPS for sample cleanup in dried blood spots (DBS) for a 7-plex LSD assay including I2S.
- Compared CIPS with ethyl acetate liquid-liquid extraction and room temperature acetonitrile precipitation.
- Analyzed extracts using optimized liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) with a 1.9-minute cycle time.
Main Results:
- CIPS combined with acetonitrile improved the detection of I2S products.
- The CIPS method demonstrated cleaner sample extracts by promoting better coagulation and separation of heme, proteins, and residual salts.
- No significant detriment to the detection of other analytes in the 6-plex assay was observed.
Conclusions:
- Cold-induced water acetonitrile phase separation (CIPS) is a promising and straightforward method for cleaner sample extracts in multiplexed LSD screening.
- This technique facilitates the development of a new 7-plex LSD screening panel, enhancing the detection of I2S for MPS-II diagnosis.
